Target Cells Research Articles

Rg3-lipo biomimetic delivery of paclitaxel enhances targeting of tumors and myeloid-derived suppressor cells.

Liposomal drug delivery systems have revolutionized traditional cytotoxic drugs. However, the relative instability and toxicity of the existing liposomal drug delivery systems compromised their efficacy. Herein, we present Rg3-lipo, an innovative drug delivery system using a glycosyl moiety-enriched ginsenoside (Rg3). This system is distinguished by its glycosyl moieties exposed on the liposomal surface. These moieties imitate human cell membranes to stabilize and evade phagocytic clearance. The Rg3-lipo system loaded with paclitaxel (PTX-Rg3-lipo) demonstrated favorable bioavailability and safety in Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys. With its glycosyl moieties recognizing tumor cells via the glucose transporter Glut1, PTX-Rg3-lipo inhibited gastric, breast, and esophageal cancers in human cancer cell lines, tumor-bearing mice, and patient-derived xenograft models. These glycosyl moieties selectively targeted myeloid-derived suppressor cells (MDSCs) through the glucose transporter Glut3 to attenuate their immunosuppressive effect. The mechanism study revealed that Rg3-lipo suppressed glycolysis and downregulated the transcription factors c-Maf and Mafb overcoming the MDSC-mediated immunosuppressive microenvironment and enhancing PTX-Rg3-lipo's antitumor effect. Taken together, we supply substantial evidence for its advantageous bioavailability and safety in multiple animal models, including nonhuman primates, and Rg3-lipo's dual targeting of cancer cells and MDSCs. Further investigation regarding Rg3-lipo's druggability will be conducted in clinical trials.

Acute Kidney Injury Following CAR-T Cell Therapy: A Nephrologist Perspective

Abstract Chimeric antigen receptor T-cell (CAR-T cell) therapy, an emerging personalized immunotherapy for various hematologic malignancies, autoimmune diseases, and other conditions, involves the modification of patients’ T cells to express a chimeric antigen receptor which recognizes tumor or autoimmune cell antigens. Thereby CAR-T cells destroy cancerous and other target cells selectively. Despite remarkable clinical improvements in patients, multiple adverse effects have been associated with CAR-T cell therapy. Among the most recognized adverse effects are cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, and tumor lysis syndrome. Even though less recognized, the incidence of acute kidney injury (AKI) ranges from 5% to 33%. The wide range of reported AKI incidence rates might depend on patient population characteristics and comorbidities and specific CAR-T cell therapy features. Even though the exact pathophysiology remains unknown, several key mechanisms including cytokine release syndrome, tumor lysis syndrome, and other factors such as direct renal toxicity of CAR-T cell therapy, conditioning regimens or other medications (e.g. antibiotics), and infectious complications (e.g. sepsis) have been proposed. Risk factors for CAR-T-related AKI include lower baseline glomerular filtration rate, higher rates of allopurinol or rasburicase use, intravenous contrast material exposure, elevated baseline lactate dehydrogenase, and grade 3 or higher cytokine release syndrome. Future prospective studies with larger patient populations are needed to gain more insight regarding the pathophysiology of CAR-T-related AKI, and, more importantly, to be able to prevent as well as to develop novel and more efficient treatment modalities. In this narrative review, we discuss the underlying pathophysiology, risk factors, potential interventions, and future directions related to AKI following CAR-T cell therapy.

Downhill running does not alter blood C1q availability or complement-dependent cytotoxicity of therapeutic monoclonal antibodies against haematological cancer cell lines in vitro.

Complement-dependent cytotoxicity (CDC) is a primary mechanism-of-action of monoclonal antibody (mAb) immunotherapies used to treat haematological cancers, including rituximab and daratumumab. However, mAb efficacy may be limited by reduced bioavailability of complement C1q - which activates the complement classical pathway following interactions with mAb-opsonised target cells. C1q is secreted by phagocytes upon recruitment to sites of muscle damage to facilitate muscular repair, hence we hypothesised that muscle damaging exercise may increase C1q 'spill-over' into blood. Additionally, other complement proteins (e.g., C1s) have been reported to increase following ultra-endurance and resistance exercise. Taken together, we hypothesised that muscle damaging exercise could be harnessed to enhance mAb-mediated CDC. In this study, n = 8 healthy males (28 ± 5-years) completed two 45-minute treadmill running protocols: (1) a flat running protocol at a speed 15% above anaerobic threshold, and (2) a downhill running protocol (- 10% slope) at the same speed. Blood samples were collected before, immediately after, and 1-hour, 24-hours, 2-days, and 4-days after exercise. Isolated serum was assessed for C1q by ELISA, and used to measure mAb (rituximab, daratumumab) mediated CDC against two haematological cancer cell lines (Raji, RPMI-8226) in vitro. Isolated plasma was assessed for markers of inflammation (C-reactive protein [CRP]), and muscle damage (creatine kinase [CK]) by turbidimetry. C1q and CDC activity were not different between running protocols and did not change over time (p > 0.05). Significantly greater perceived muscle soreness (p < 0.001) and fluctuations observed from baseline to 24-hours post-exercise in the downhill running trial in CK (+ 171%) and CRP (+ 66%) suggests some degree of muscle damage was present. It is possible that any increase in C1q post-exercise may have been masked by the increase and subsequent interaction with CRP, which utilises C1q to facilitate muscular repair. This is the first study to investigate whether exercise can increase circulating C1q and improve mAb-mediated CDC and our findings show that downhill running exercise does not increase circulating C1q nor improve CDC in vitro.

The Immunologic Downsides Associated with the Powerful Translation of Current COVID-19 Vaccine mRNA Can Be Overcome by Mucosal Vaccines

The action of mRNA-based vaccines requires the expression of the antigen in cells targeted by lipid nanoparticle–mRNA complexes. When the vaccine antigen is not fully retained by the producer cells, its local and systemic diffusion can have consequences depending on both the levels of antigen expression and its biological activity. A peculiarity of mRNA-based COVID-19 vaccines is the extraordinarily high amounts of the Spike antigen expressed by the target cells. In addition, vaccine Spike can be shed and bind to ACE-2 cell receptors, thereby inducing responses of pathogenetic significance including the release of soluble factors which, in turn, can dysregulate key immunologic processes. Moreover, the circulatory immune responses triggered by the vaccine Spike is quite powerful, and can lead to effective anti-Spike antibody cross-binding, as well as to the emergence of both auto- and anti-idiotype antibodies. In this paper, the immunologic downsides of the strong efficiency of the translation of the mRNA associated with COVID-19 vaccines are discussed together with the arguments supporting the idea that most of them can be avoided with the advent of next-generation, mucosal COVID-19 vaccines.

Abstract B025: Redox reprogramming by SOD1 knockdown: A novel liposomal siRNA strategy to overcome platinum resistance in ovarian cancer

Abstract Platinum resistance remains a significant obstacle in the treatment of advanced-stage ovarian cancer, frequently leading to therapeutic failure. Our previous research identified Superoxide Dismutase 1 (SOD1) as a key antioxidant target for re-sensitizing platinum-resistant ovarian cancer cells to platinum-based therapies through both enzymatic inhibition and RNA interference (RNAi) utilizing a graphene-based siRNA delivery platform. Building on these findings, we have now developed a liposomal formulation of chemically modified siRNA targeting SOD1, which demonstrates potent knockdown efficiency and significantly enhances the sensitivity of resistant ovarian cancer cells to cisplatin in vivo. While SOD1 inhibition or knockdown via RNAi effectively re-sensitizes resistant cells to cisplatin, the underlying redox-sensitive signaling pathways remain largely unexplored in ovarian cancer. Our current study reveals that SOD1 knockdown acts as a redox switch, triggering cellular reprogramming in drug-resistant cells through post-translational cysteine modifications. This reprogramming leads to the rewiring of critical cancer hallmarks, culminating in the restoration of a cisplatin-sensitive phenotype. These findings suggest that a liposomal, chemically modified SOD1 siRNA formulation could serve as a promising therapeutic approach for reversing platinum resistance in ovarian cancer by exploiting redox-mediated reprogramming of cancer hallmarks. This study not only underscores the therapeutic potential of targeting SOD1 but also highlights the broader implications of redox signaling in the etiology of platinum resistance, providing a novel avenue for therapeutic intervention. Citation Format: Attila Szenasi, Sonia Rocha, Mu Wang. Redox reprogramming by SOD1 knockdown: A novel liposomal siRNA strategy to overcome platinum resistance in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B025.

A systematic overview of strategies for photosensitizer and light delivery in antibacterial photodynamic therapy for lung infections.

Antimicrobial photodynamic therapy (aPDT) emerges as a viable treatment strategy for infections resistant to conventional antibiotics. A complex interplay of factors, including intracellular photosensitizer (PS) accumulation, photochemical reaction type, and oxygen levels, determines the efficacy of aPDT. Recent progress includes the development of modified PSs with enhanced lipophilicity and target-specific strategies to improve bacterial cell wall penetration and targeting. Nanotechnology-based approaches, such as using nanomaterials for targeted PS delivery, have shown promise in enhancing aPDT efficacy. Advancements in light delivery methods for aPDT, such as transillumination of large lesions and local light delivery using fiber optic techniques, are also being explored to optimize treatment efficacy in clinical settings. The limited number of animal models and clinical trials specifically designed to assess the efficacy of aPDT for lung infections highlights the need for further research in this critical area. The potential prospects of aPDT for lung tissue infections originating from antibiotic-resistant bacterial infections are also discussed in this review.

Recent advances in polymeric and lipid stimuli-responsive nanocarriers for cell-based cancer immunotherapy.

Conventional cancer therapy has major limitations, including non-specificity, unavoidable side effects, low specific tumor accumulation and systemic toxicity. In recent years, more effective and precise treatment methods have been developed, including cell-based immunotherapy. Carriers that can accurately and specifically target cells and equip them to combat cancer cells are particularly important for developing this therapy. As a result, attention has been drawn to smart nanocarriers that can react to specific stimuli. Thus, stimuli-responsive nanocarriers have attracted increasing attention because they can change their physicochemical properties in response to stimulus conditions, such as pH, enzymes, redox agents, hypoxia, light and temperature. This review highlights recent advances in various stimuli-responsive nanocarriers, discussing loading, targeted delivery, cellular uptake, biocompatibility and immunomodulation in cell-based immunotherapy. Finally, future challenges and perspectives regarding the possible clinical translation of nanocarriers are discussed.

Optical control of cardiac electrophysiology by the photochromic ligand azobupivacaine 2.

Patients suffering from ischaemic heart disease and heart failure are at high risk of recurrent ventricular arrhythmias (VAs), eventually leading to sudden cardiac death. While high-voltage shocks delivered by an implantable defibrillator may prevent sudden cardiac death, these interventions themselves impair quality of life and raise both morbidity and mortality, which accentuates the need for developing novel defibrillation techniques. Photopharmacology allows for reversible control of biological processes by light. When relying on synthetic and externally applied chromophores, it renders genetic modification of target cells dispensable and may hence be advantageous over optogenetic approaches. Here, the photochromic ligand azobupivacaine 2 (AB2) was probed as a modulator of cardiac electrophysiology in an ex vivo intact mouse heart model. By reversibly blocking voltage-gated Na+ and K+ channels, photoswitching of AB2 modulated both the ventricular effective refractory period and the conduction velocity in native heart tissue. Moreover, photoswitching of AB2 was able to convert VA into sinus rhythm. The present study provides the first proof of concept that AB2 enables gradual control of cardiac electrophysiology by light. AB2 may hence open the door to the development of an optical defibrillator based on photopharmacology.

Mannose/stearyl chloride doubly functionalized polyethylenimine as a nucleic acid vaccine carrier to promote macrophage uptake

Transmembrane transport remains a significant challenge for nucleic acid vaccine vectors. Promoting the ability of immune cells, such as macrophages, to capture foreign stimuli is also an effective approach to improving cross-presentation. In addition, polyethyleneimine (PEI) has gained attention in the field of nucleic acid vaccine carriers due to its excellent gene transfection efficiency and unique proton buffering effect. However, although high molecular weight PEI exhibits high efficiency, its high-density positive charges make it highly toxic, which limits its application. In this study, mannose/stearyl chloride functionalized polyethylenimine (SA-Man-PEI) was prepared by functionalizing PEI (molecular weight of 25 kDa) with mannose with immunomodulatory and phagocyte targeting effects, and an alkyl hydrophobic chain segment, which could easily promote cell uptake. Moreover, the functionalized-PEI retains a strong proton buffering effect, which helps the carrier escape from the lysosome. The particle sizes of the composite particles formed by SA-Man-PEI and ovalbumin (OVA) were below 200 nm, with good storage stability at both 4 °C and 37 °C. At a drug concentration of 2 μg/mL, the cell survival rate of functionalized-PEI was 19.2% higher than that of unfunctionalized PEI. In vitro macrophage endocytosis experiments showed that SA-Man-PEI could significantly enhance the macrophage uptake of composite particles, compared to unfunctionalized PEI or single-functionalized PEI. This study offers a new approach for developing PEI as a nucleic acid vaccine carrier, which could simultaneously enhance cell targeting and promote cell uptake.

Possible Mechanisms of the Influence of Oxytocin and Vazopressin on Perception and Memory of Odors and on Social Behavior

A possible mechanism is proposed for the influence of oxytocin and vasopressin on the functioning of the neural network in the CNS, in which olfactory information is processed and stored, and which plays an important role in social behavior. The effect of these neuropeptides on postsynaptic receptors associated with Gq/11 proteins contributes to the induction of LTP of the efficacy of excitatory synaptic inputs to the main projection cells and to inhibitory interneurons in the prefrontal cortex, hippocampus, piriform cortex, anterior olfactory nucleus, olfactory bulb and nucleus accumbens, including the olfactory tubercle. As a result of disynaptic inhibition in each of the structures, the signal-to-noise ratio is improved and the transmission of strong signals through projection neurons to their target cells is facilitated. Due to the fact, that oxytocin promotes the release of dopamine by the neurons of the ventral tegmental area, the conditions for processing and memorizing olfactory information in the interconnected olfactory and hippocampal neural networks, including cortical and subcortical structures, are improved, and attention is also included in this processing. Long-term modification of the effectiveness of interneuronal connections in these networks under the influence of oxytocin and dopamine contributes to the formation and stabilization of contrasting neuronal representation of odors formed in cortical areas. Orientation of attention increases the significance of socially important olfactory stimuli and improves the conditions for the functioning of the reinforcement system necessary for adequate social behavior. Taking into account the known data on the correlation between social behavior and the density of oxytocin and vasopressin receptors on neurons of different structures, understanding the mechanisms of the influence of these neuropeptides on the functioning of the olfactory system can be useful for finding ways to correct behavior if necessary.

Implications of glioblastoma-derived exosomes in modifying the immune system: state-of-the-art and challenges.

Glioblastoma is a brain cancer with a poor prognosis. Failure of classical chemotherapy and surgical treatments indicates that new therapeutic approaches are needed. Among cell-free options, exosomes are versatile extracellular vesicles (EVs) that carry important cargo across barriers suchas the blood-brain barrier (BBB) to their target cells. Thismakes exosomes an interesting option for the treatment ofglioblastoma. Moreover, exosomes can comprise many therapeutic cargos, including lipids, proteins, and nucleic acids,sampled from special intercellular compartments of their origin cell. Cells exposed to various immunomodulatory stimulican generate exosomes enriched in specific therapeutic molecules. Notably, the secretion of exosomes could modify theimmune response in innate and adaptive immune systems. For instance, glioblastoma-associated exosomes (GBex) uptake bymacrophages could influence macrophage dynamics (e.g., shifting CD markers expression). Expression of critical immunoregulatory proteins such as cytotoxic T-lymphocyte antigen-1 (CTLA1) and programmed death-1 (PD-1) on GBex indicates the direct crosstalk of these nano-size vesicles with the immune system. The present study reviews the role of exosomes in immune system cells, including Bcells, Tcells, natural killer (NK) cells, and dendritic cells (DCs), as well as novel technologies in the field.

KAT6B is required for histone 3 lysine 9 acetylation and SOX gene expression in the developing brain.

Heterozygous mutations in the histone lysine acetyltransferase gene KAT6B (MYST4/MORF/QKF) underlie neurodevelopmental disorders, but the mechanistic roles of KAT6B remain poorly understood. Here, we show that loss of KAT6B in embryonic neural stem and progenitor cells (NSPCs) impaired cell proliferation, neuronal differentiation, and neurite outgrowth. Mechanistically, loss of KAT6B resulted in reduced acetylation at histone H3 lysine 9 and reduced expression of key nervous system development genes in NSPCs and the developing cortex, including the SOX gene family, in particular Sox2, which is a key driver of neural progenitor proliferation, multipotency and brain development. In the fetal cortex, KAT6B occupied the Sox2 locus. Loss of KAT6B caused a reduction in Sox2 promoter activity in NSPCs. Sox2 overexpression partially rescued the proliferative defect of Kat6b -/- NSPCs. Collectively, these results elucidate molecular requirements for KAT6B in brain development and identify key KAT6B targets in neural precursor cells and the developing brain.

Nutrient transporter pattern in CD56dim NK cells: CD16 (FcγRIIIA)-dependent modulation and association with memory NK cell functional profile

BackgroundHuman memory NK cells represent a heterogeneous CD56dim population that expands and persists in human cytomegalovirus (HCMV)-seropositive healthy individuals. They are characterized by the preferential, not fully overlapping, expression of NKG2C (activating receptor for HLA-E) and CD57 maturation marker, and by the lack of FcεRIγ adaptor chain. Hyperresponsiveness to Fcγ receptor IIIA (CD16) engagement represents the distinctive functional signature of memory NK cells. Although CD16 engagement was shown to acutely enhance glycolytic and oxidative pathways, its capability to induce a persisting metabolic reprogramming of human NK cells is poorly understood yet.ResultsHere, we describe the peculiar nutrient transporter expression pattern of FcεRIγ- memory NK cells, characterized by higher levels of CD98 neutral amino acid antiporter and CD71 transferrin receptor, and lower expression of GLUT1 glucose transporter, with respect to FcεRIγ+ conventional NK cells. Although CD16 engagement acutely enhances glycolytic and oxidative pathways, its capability to induce a persisting metabolic reprogramming of human NK cells is poorly understood yet. Our results firstly show that sustained CD16 engagement by contact with IgG-opsonized target cells induces the mTORC1-dependent upregulation of CD98 and CD71 nutrient receptors on CD56dim NK cells, in a transporter-specific fashion, that is finely tuned by cell-dependent (grade of functional maturation, and memory or conventional lineage) and stimulus-dependent (time length and cooperation with cytokines) factors. We also demonstrate that CD98 antiporter function is required for CD16-dependent IFN-γ production, and that enhanced CD98-mediated neutral amino acid uptake associates with heightened memory NK cell functional response.ConclusionCollectively, our work documents that CD16 engagement leads to a metabolic rewiring of human NK cells and suggests that a distinct nutrient transporter expression pattern may contribute to memory NK cell peculiar functional features.

Induction of ectosome formation by binding of phospholipases D from Loxosceles venoms to endothelial cell surface: Mechanism of interaction.

Members of the phospholipase D (PLD) superfamily found in Loxosceles spider venoms are potent toxins with inflammatory and necrotizing activities. They degrade phospholipids in cell membranes, generating bioactive molecules that activate skin cells. These skin cells, in turn, activate leukocytes involved in dermonecrosis, characterized by aseptic coagulative necrosis. Although the literature has advanced in understanding the structure-function relationship, the cell biology resulting from the interactions of these molecules with cells remains poorly understood. In this study, we show that different cells exposed to recombinant PLDs bind these molecules to their plasma membrane, leading to the subsequent organization of extracellular microvesicles/ectosomes. The binding occurs as quickly as five minutes or less after exposure, increases over time, and eventually, the PLDs are expelled from the cell surface without generating cytotoxicity. PLDs are not endocytosed, nor do they spatially colocalize with acidic organelles in the intracellular environment. At least two regions of PLDs - the domain involved in magnesium ion coordination and the choline binding site - appear to play a role in cell surface binding and ectosome organization. However, the amino acids involved in catalysis do not participate in these events. The binding of these PLDs to the cell membrane, independent of catalytic activity, is sufficient to trigger intracellular signaling and enhance the expression of the pro-inflammatory IL-8 gene. These results are supported by the observation that isoforms of PLDs lacking catalytic activity induce an inflammatory response in vivo when injected into the skin of rabbits, without causing dermonecrosis. Our data indicate that these PLDs bind to the surface of target cells, promoting the organization of extracellular vesicles/ectosomes. Subsequently, these events activate pro-inflammatory genes and induce an inflammatory response in vivo. The binding to cells is not dependent on amino acids involved in catalysis but rather on amino acids involved in magnesium coordination. The binding of PLDs to the cell surface, formation of ectosomes, and activation of cells appear to initiate signals involved in inflammatory responses that can lead to dermonecrosis in accidents. This correlation is supported by experimental observations indicating that the events of toxin binding to cells, formation of microvesicles, and inflammatory responses observed both in vitro and in vivo are interconnected.

Granzyme mRNA-miRNA interaction and its implication to functional impact.

Granzyme activity can affect the processing and stability of miRNAs within target cells. They also could induce changes in miRNA expression that impact apoptotic signaling. Granzyme-induced apoptosis might result in changes to the miRNA profile, which can further influence the apoptosis and inflammation processes. The aim of this study was to bioinformatically analyze which miRNAs and transcription factors bind to the CDS and UTR regions of the granzyme family to regulate gene expression in relation to granzyme evolution and their association with human cancer diseases. The expression patterns of granzyme genes were analyzed in various human tissues. MiRNAs binding to the CDS and UTR of the granzyme family were examined, and the transcription factors binding to these miRNAs binding sites were also analyzed. Cytoscape program was used to visualize and analyze the networks of interactions between granzyme mRNA and miRNAs. Additionally, the evolutionary patterns of the granzyme family in relation to miRNAs and transcription factors binding were investigated. Analysis of the expression patterns of the granzyme family in various human tissues shows that GZMA and GZMK are strongly expressed in lymph nodes. GZMB exhibits strong expression in the bone marrow, while GZMA is prominently expressed in the spleen. Twenty-two miRNAs bind to both GZMK and GZMB mRNA, while six miRNAs bind to both GZMK and GZMM mRNA. The only miRNA that binds to GZMK, GZMB, GZMM, and GZMA mRNA is hsa-miR-146a-5p. Transcription factors JUND, FOS, and JUN are distinctly interconnected with has-miR-5696 and GZMK. Association data between the granzyme family and cancers showed that various miRNAs were consistently implicated and exhibited either upregulation or downregulation. Although the granzyme family possesses distinct genetic information, it shows relatively high expression levels in the lymph node, spleen, and bone marrow. Many miRNAs specifically regulate granzyme gene expression, and various transcription factors are involved. Analyzing the granzyme genes-miRNAs-transcription factors-related network will provide crucial insights into the mechanisms of cancer development and suppression.

Designed Cell-Penetrating Peptide Constructs for Inhibition of Pathogenic Protein Self-Assembly

Peptides possess a number of pharmacologically desirable properties, including greater chemical diversity than other biomolecule classes and the ability to selectively bind to specific targets with high potency, as well as biocompatibility, biodegradability, and ease and low cost of production. Consequently, there has been considerable interest in developing peptide-based therapeutics, including amyloid inhibitors. However, a major hindrance to the successful therapeutic application of peptides is their poor delivery to target tissues, cells or subcellular organelles. To overcome these issues, recent efforts have focused on engineering cell-penetrating peptide (CPP) antagonists of amyloidogenesis, which combine the attractive intrinsic properties of peptides with potent therapeutic effects (i.e., inhibition of amyloid formation and the associated cytotoxicity) and highly efficient delivery (to target tissue, cells, and organelles). This review highlights some promising CPP constructs designed to target amyloid aggregation associated with a diverse range of disorders, including Alzheimer’s disease, transmissible spongiform encephalopathies (or prion diseases), Parkinson’s disease, and cancer.

In utero human cytomegalovirus infection expands NK-like FcγRIII+ CD8+ T cells that mediate Fc antibody functions.

Human cytomegalovirus (HCMV) profoundly impacts host T and natural killer (NK) cells across the lifespan, yet how this common congenital infection modulates developing fetal immune cell compartments remains underexplored. Using cord blood from neonates with and without congenital HCMV (cCMV) infection, we identify an expansion of Fcγ receptor III (FcγRIII)-expressing CD8+ T cells following HCMV exposure in utero. Most FcγRIII+ CD8+ T cells express the canonical αβ T cell receptor (TCR) but a proportion express non-canonical γδ TCR. FcγRIII+ CD8+ T cells are highly differentiated and have increased expression of NK cell markers and cytolytic molecules. Transcriptional analysis reveals FcγRIII+ CD8+ T cells upregulate T-bet and downregulate BCL11B, known transcription factors that govern T/NK cell fate. We show that FcγRIII+ CD8+ T cells mediate antibody-dependent IFNγ production and degranulation against IgG-opsonized target cells, similar to NK cell antibody-dependent cellular cytotoxicity (ADCC). FcγRIII+ CD8+ T cell Fc effector functions were further enhanced by interleukin-15 (IL-15), as has been observed in neonatal NK cells. Our study reveals that FcγRIII+ CD8+ T cells elicited in utero by HCMV infection can execute Fc-mediated effector functions bridging cellular and humoral immunity and may be a promising target for antibody-based therapeutics and vaccination in early life.

EXTH-71. A MECHANISTIC INVESTIGATION INTO AUGER THERAPY: 2-DIMENSIONAL THIN-FILMS TO CHARACTERIZE THERAPEUTIC RANGEIN VITRO

Abstract Metallic nanoparticles can improve the accuracy and effectiveness of conventional radiotherapy in the treatment of solid tumors through a light-matter interaction known as the Auger effect. In this work, we present the assembly of an iron oxide (Fe3O4) nanoparticle (NPs) layer within a biocompatible polyelectrolyte film using ionic self-assembly to enable the spatial separation of NPs from their target cells. Films exhibited uniform nanoparticle coverage as well as strong stability in cell culture conditions. To investigate the applicability of NMDR with non-internalized NPs, conditions with (NP-PEM) and without (PEM) embedded nanoparticles were used to evaluate the therapeutic effect of physically separated nanoparticles on cells cultured on the film surface via a measure of intracellular reactive oxygen (ROS) generation and histone phosphorylation (yH2AX) after irradiation from a 137Cesium source. Cells cultured on NP-PEM demonstrated a 78.4% increase in ROS generation when compared to cells cultured on PEM films. When adjusting for cell count, NP-PEM films induce 70.5% greater ROS generation. In investigating nanoparticle-mediated deposition of radiation (NMDR), these films allow for the determination of the extent of the therapeutic effect when NPs are not internalized into their targeted cells, potentially providing indications with respect to off-target effects. Cells were irradiated on films with nanoparticle distances from 25.9 nm to 97.5 nm to interrogate the NMDR effect size at a range of distances. yH2AX phosphorylation exhibited an exponential decay until 69.8 nm, where there was no difference when compared to nanoparticle-free films. This work highlights the ability of iron oxide NPs to deliver NMDR at a known, quantifiable distance, providing insights for further translational applications.

MiR-630 as a therapeutic target in pancreatic cancer stem cells: modulation of the PRKCI-Hedgehog signaling axis.

MicroRNAs (miRNAs) are critical regulators of cancer progression, prompting our investigation into the specific function of miR-630 in pancreatic cancer stem cells (PCSCs). Analysis of miRNA and mRNA expression data in PCSCs revealed downregulation of miR-630 and upregulation of PRKCI, implying a potential role for miR-630 in PCSC function and tumorigenicity. Functional assays confirmed that miR-630 directly targets PRKCI, leading to the suppression of the Hedgehog signaling pathway and consequent inhibition of PCSC self-renewal and tumorigenicity in murine models. This study unveiled the modulation of the PRKCI-Hedgehog signaling axis by miR-630, highlighting its promising therapeutic potential for pancreatic cancer (PC) treatment. MiR-630 emerges as a pivotal regulator in PCSC biology, opening up new avenues for targeted interventions in PC. The inhibitory effect of miR-630 on PCSC behavior underscores its potential as a valuable therapeutic target, offering insights into innovative treatment strategies for this challenging disease.

DNAR-11. INHIBITION OF RAD52 ACTIVITY INDUCES DNA DAMAGE BY ACCUMULATING R-LOOPS IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma (DMG) is one of the most devastating childhood cancers with a median survival of &amp;lt; 1year from diagnosis. There is a critical need for new therapeutics for improving treatment outcomes for DMG patients. We performed genome-wide CRISPR/Cas9 screening and identified RAD52 as a potential therapeutic target in DMG cells. RAD52 inhibition, using shRNA-mediated RAD52 depletion and treatment with RAD52 inhibitor, D-I03, suppressed DMG cell proliferation. Western blotting revealed that RAD52 inhibition increased DNA double-starand breaks (DSBs) marker γH2AX while it decreased repair markers BRCA1 and RAD51. Also, RAD52 inhibition causes a sustained level of phosphorylated RAD50 and γH2AX in irradiated DMG cells over 24 hours. Immunocytochemistry (ICC) of γH2AX and repair marker 53BP1 showed that RAD52 inhibition sustained DNA damage with high levels of γH2AX at 24 hours following radiation while the level of 53BPI was decreased, thereby inhibiting DNA DSB repair. DNA repair assay showed that RAD52 inhibition suppressed DNA DSB repair through a homologous recombination pathway. RNA sequencing showed that RAD52 inhibition downregulated genes associated with the DNA repair pathway. Importantly, RAD52 inhibition increased the radiosensitivity of DMG cells. To understand the mechanism of RAD52 inhibition on DNA damage, we performed dot plot assay and ICC of S9.6 which recognizes DNA-RNA hybrids (also known as R-loops) and found accumulation of R-loop formation in DMG cells treated with RAD52 inhibitor. In addition, RAD52 inhibition decreased the expression of RNA polymerase II. Finally, the combination therapy of RAD52 inhibitor and radiation inhibited tumor growth and increased survival of mice bearing DMG patient-derived xenografts, outperforming either monotherapy. Together, our results highlight that RAD52 inhibition induces DNA damage by accumulating R-loops, results in reducing DMG cell proliferation, while also increase DMG radiosensitivty, and providing a rationale for developing combination therapy with radiation for the treatment of DMG.