Danger Signals Research Articles

Programmable melanoma-targeted radio-immunotherapy via fusogenic liposomes functionalized with multivariate-gated aptamer assemblies

Radio-immunotherapy exploits the immunostimulatory features of ionizing radiation (IR) to enhance antitumor effects and offers emerging opportunities for treating invasive tumor indications such as melanoma. However, insufficient dose deposition and immunosuppressive microenvironment (TME) of solid tumors limit its efficacy. Here we report a programmable sequential therapeutic strategy based on multifunctional fusogenic liposomes (Lip@AUR-ACP-aptPD-L1) to overcome the intrinsic radio-immunotherapeutic resistance of solid tumors. Specifically, fusogenic liposomes are loaded with gold-containing Auranofin (AUR) and inserted with multivariate-gated aptamer assemblies (ACP) and PD-L1 aptamers in the lipid membrane, potentiating melanoma-targeted AUR delivery while transferring ACP onto cell surface through selective membrane fusion. AUR amplifies IR-induced immunogenic death of melanoma cells to release antigens and damage-associated molecular patterns such as adenosine triphosphate (ATP) for triggering adaptive antitumor immunity. AUR-sensitized radiotherapy also upregulates matrix metalloproteinase-2 (MMP-2) expression that combined with released ATP to activate ACP through an “and” logic operation-like process (AND-gate), thus triggering the in-situ release of engineered cytosine-phosphate-guanine aptamer-based immunoadjuvants (eCpG) for stimulating dendritic cell-mediated T cell priming. Furthermore, AUR inhibits tumor-intrinsic vascular endothelial growth factor signaling to suppress infiltration of immunosuppressive cells for fostering an anti-tumorigenic TME. This study offers an approach for solid tumor treatment in the clinics.

A pyroptosis-enhanced leucocyte-hitchhiking liposomal nanoplatform for potentiated immunotherapy of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a highly malignant tumor with unsatisfactory response to immunotherapy. Pyroptosis, a recently discovered form of regulated cell death (RCD), possesses a huge potential to enhance the immunotherapy efficiency against HCC. To achieve efficient drug delivery and ideal activation of antitumor immunity, an E-selectin modified liposomal nanoplatform co-loading gemcitabine elaidate and BMS-202 (a small molecule programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitor) was designed. Following intravenous injection, the liposomal nanoplatform could efficiently bind to sialylated carbohydrates on the surface of peripheral blood leucocytes via E-selectin, subsequently hitchhiking with leucocytes to realize substantial accumulation in the HCC tissue. After cellular uptake by HCC cells, the released gemcitabine could trigger gasdermin E (GSDME)-dependent pyroptosis with the release of danger-associated molecular patterns (DAMPs) and pro-inflammatory cytokines, thus generating antitumor immunity. The released BMS-202 could further relieve immune suppression by blocking the formation of PD-1/PD-L1 complex. More importantly, gemcitabine-triggered tumor pyroptosis enhanced natural orientation of leucocytes to inflammatory tumor site, further increasing the nanoplatform delivery by facilitating tumor leucocyte infiltration through a positive feedback loop. The in vivo efficacy of the fabricated liposomes demonstrated a favorable antitumor immunity by promoting dendritic cell maturation and T cell activation. In summary, this pyroptosis-enhanced leucocyte-hitchhiking liposomal nanoplatform suggests synergistic antitumor activity and unique ability to modulate drug delivery, showing promise as a highly efficient strategy for potentiated tumor immunotherapy, with a potential for clinical translation.

Beyond the danger signal: RNA aggregates orchestrate immunotherapy

Mendez-Gomez et al. recently demonstrated the transformative potential of RNA-lipid particle aggregates (RNA-LPAs) in immunotherapy. By reprogramming the tumor microenvironment (TME) and potentiating antitumor immunity, RNA-LPAs target primary tumors and elicit robust systemic immunity. This innovative platform holds promise for translating preclinical success into tangible clinical benefits.

Double-Edged Sword: Exploring the Mitochondria-Complement Bidirectional Connection in Cellular Response and Disease.

Mitochondria serve an ultimate purpose that seeks to balance the life and death of cells, a role that extends well beyond the tissue and organ systems to impact not only normal physiology but also the pathogenesis of diverse diseases. Theorized to have originated from ancient proto-bacteria, mitochondria share similarities with bacterial cells, including their own circular DNA, double-membrane structures, and fission dynamics. It is no surprise, then, that mitochondria interact with a bacterium-targeting immune pathway known as a complement system. The complement system is an ancient and sophisticated arm of the immune response that serves as the body's first line of defense against microbial invaders. It operates through a complex cascade of protein activations, rapidly identifying and neutralizing pathogens, and even aiding in the clearance of damaged cells and immune complexes. This dynamic system, intertwining innate and adaptive immunity, holds secrets to understanding numerous diseases. In this review, we explore the bidirectional interplay between mitochondrial dysfunction and the complement system through the release of mitochondrial damage-associated molecular patterns. Additionally, we explore several mitochondria- and complement-related diseases and the potential for new therapeutic strategies.

Necroptosis stimulates interferon-mediated protective anti-tumor immunity

Necroptosis is an inflammatory form of cell suicide that critically depends on the kinase activity of Receptor Interacting Protein Kinase 3 (RIPK3). Previous studies showed that immunization with necroptotic cells conferred protection against subsequent tumor challenge. Since RIPK3 can also promote apoptosis and NF-κB-dependent inflammation, it remains difficult to determine the contribution of necroptosis-associated release of damage-associated molecular patterns (DAMPs) in anti-tumor immunity. Here, we describe a system that allows us to selectively induce RIPK3-dependent necroptosis or apoptosis with minimal NF-κB-dependent inflammatory cytokine expression. In a syngeneic tumor challenge model, immunization with necroptotic cells conferred superior protection against subsequent tumor challenge. Surprisingly, this protective effect required CD4+ T cells rather than CD8+ T cells and is dependent on host type I interferon signaling. Our results provide evidence that death-dependent type I interferon production following necroptosis is sufficient to elicit protective anti-tumor immunity.

A Dynamic Protocol to Explore NLRP3 Inflammasome Activation in Cerebral Organoids.

The NLRP3 inflammasome plays a crucial role in the inflammatory response, reacting to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). This response is essential for combating infections and restoring tissue homeostasis. However, chronic activation can lead to detrimental effects, particularly in neuropsychiatric and neurodegenerative diseases. Our study seeks to provide a method to effectively measure the NLRP3 inflammasome's activation within cerebral organoids (COs), providing insights into the underlying pathophysiology of these conditions and enabling future studies to investigate the development of targeted therapies.

Repair of the Infarcted Heart: Cellular Effectors, Molecular Mechanisms and Therapeutic Opportunities.

The adult mammalian heart has limited endogenous regenerative capacity and heals through the activation of inflammatory and fibrogenic cascades that ultimately result in the formation of a scar. After infarction, massive cardiomyocyte death releases a broad range of damage-associated molecular patterns that initiate both myocardial and systemic inflammatory responses. TLRs (toll-like receptors) and NLRs (NOD-like receptors) recognize damage-associated molecular patterns (DAMPs) and transduce downstream proinflammatory signals, leading to upregulation of cytokines (such as interleukin-1, TNF-α [tumor necrosis factor-α], and interleukin-6) and chemokines (such as CCL2 [CC chemokine ligand 2]) and recruitment of neutrophils, monocytes, and lymphocytes. Expansion and diversification of cardiac macrophages in the infarcted heart play a major role in the clearance of the infarct from dead cells and the subsequent stimulation of reparative pathways. Efferocytosis triggers the induction and release of anti-inflammatory mediators that restrain the inflammatory reaction and set the stage for the activation of reparative fibroblasts and vascular cells. Growth factor-mediated pathways, neurohumoral cascades, and matricellular proteins deposited in the provisional matrix stimulate fibroblast activation and proliferation and myofibroblast conversion. Deposition of a well-organized collagen-based extracellular matrix network protects the heart from catastrophic rupture and attenuates ventricular dilation. Scar maturation requires stimulation of endogenous signals that inhibit fibroblast activity and prevent excessive fibrosis. Moreover, in the mature scar, infarct neovessels acquire a mural cell coat that contributes to the stabilization of the microvascular network. Excessive, prolonged, or dysregulated inflammatory or fibrogenic cascades accentuate adverse remodeling and dysfunction. Moreover, inflammatory leukocytes and fibroblasts can contribute to arrhythmogenesis. Inflammatory and fibrogenic pathways may be promising therapeutic targets to attenuate heart failure progression and inhibit arrhythmia generation in patients surviving myocardial infarction.

Toll-like receptors in breast cancer immunity and immunotherapy.

Toll-like receptors (TLRs) are a key family of pattern recognition receptors (PRRs) in the innate immune system. The activation of TLRs will not only prevent pathogen infection but also respond to damage-induced danger signaling. Increasing evidence suggests that TLRs play a critical role in breast cancer development and treatment. However, the activation of TLRs is a double-edged sword that can induce either pro-tumor activity or anti-tumor effect. The underlying mechanisms of these opposite effects of TLR signaling in cancer are not fully understood. Targeting TLRs is a promising strategy for improving breast cancer treatment, either as monotherapies or by improving other current therapies. Here we provide an update on the role of TLRs in breast cancer immunity and immunotherapy.

Cytosolic Escape of Mitochondrial DNA Triggers cGAS-STING Pathway-Dependent Neuronal PANoptosis in Response to Intermittent Hypoxia.

Intermittent hypoxia (IH) is the predominant pathophysiological disturbance in obstructive sleep apnea (OSA), characterized by neuronal cell death and neurocognitive impairment. We focus on the accumulated mitochondrial DNA (mtDNA) in the cytosol, which acts as a damage-associated molecular pattern (DAMP) and activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, a known trigger for immune responses and neuronal death in degenerative diseases. However, the specific role and mechanism of the mtDNA-cGAS-STING axis in IH-induced neural damage remain largely unexplored. Here, we investigated the involvement of PANoptosis, a novel type of programmed cell death linked to cytosolic mtDNA accumulation and the cGAS-STING pathway activation, in neuronal cell death induced by IH. Our study found that PANoptosis occurred in primary cultures of hippocampal neurons and HT22 cell lines exposed to IH. In addition, we discovered that during IH, mtDNA released into the cytoplasm via the mitochondrial permeability transition pore (mPTP) activates the cGAS-STING pathway, exacerbating PANoptosis-associated neuronal death. Pharmacologically inhibiting mPTP opening or depleting mtDNA significantly reduced cGAS-STING pathway activation and PANoptosis in HT22 cells under IH. Moreover, our findings indicated that the cGAS-STING pathway primarily promotes PANoptosis by modulating endoplasmic reticulum (ER) stress. Inhibiting or silencing the cGAS-STING pathway substantially reduced ER stress-mediated neuronal death and PANoptosis, while lentivirus-mediated STING overexpression exacerbated these effects. In summary, our study elucidates that cytosolic escape of mtDNA triggers cGAS-STING pathway-dependent neuronal PANoptosis in response to IH, mainly through regulating ER stress. The discovery of the novel mechanism provides theoretical support for the prevention and treatment of neuronal damage and cognitive impairment in patients with OSA.

Comparative RNA-seq analysis of Arabidopsis thaliana response to AtPep1 and flg22, reveals the identification of PP2-B13 and ACLP1 as new members in pattern-triggered immunity.

In this research, a high-throughput RNA sequencing-based transcriptome analysis technique (RNA-Seq) was used to evaluate differentially expressed genes (DEGs) in the wild type Arabidopsis seedlings in response to AtPep1, a well-known peptide representing an endogenous damage-associated molecular pattern (DAMP), and flg22, a well-known microbe-associated molecular pattern (MAMP). We compared and dissected the global transcriptional landscape of Arabidopsis thaliana in response to AtPep1 and flg22 and could identify shared and unique DEGs in response to these elicitors. We found that while a remarkable number of flg22 up-regulated genes were also induced by AtPep1, 256 genes were exclusively up-regulated in response to flg22, and 328 were exclusively up-regulated in response to AtPep1. Furthermore, among down-regulated DEGs upon flg22 treatment, 107 genes were exclusively down-regulated by flg22 treatment, while 411 genes were exclusively down-regulated by AtPep1. We found a number of hitherto overlooked genes to be induced upon treatment with either flg22 or with AtPep1, indicating their possible involvement general pathways in innate immunity. Here, we characterized two of them, namely PP2-B13 and ACLP1. pp2-b13 and aclp1 mutants showed increased susceptibility to infection by the virulent pathogen Pseudomonas syringae DC3000 and its mutant Pst DC3000 hrcC (lacking the type III secretion system), as evidenced by increased proliferation of the two pathogens in planta. Further, we present evidence that the aclp1 mutant is deficient in ethylene production upon flg22 treatment, while the pp2-b13 mutant is deficient in the production of reactive oxygen species (ROS). The results from this research provide new information for a better understanding of the immune system in Arabidopsis.

Exploiting cell death and tumor immunity in cancer therapy: challenges and future directions.

Cancer remains a significant global challenge, with escalating incidence rates and a substantial burden on healthcare systems worldwide. Herein, we present an in-depth exploration of the intricate interplay between cancer cell death pathways and tumor immunity within the tumor microenvironment (TME). We begin by elucidating the epidemiological landscape of cancer, highlighting its pervasive impact on premature mortality and the pronounced burden in regions such as Asia and Africa. Our analysis centers on the pivotal concept of immunogenic cell death (ICD), whereby cancer cells succumbing to specific stimuli undergo a transformation that elicits robust anti-tumor immune responses. We scrutinize the mechanisms underpinning ICD induction, emphasizing the release of damage-associated molecular patterns (DAMPs) and tumor-associated antigens (TAAs) as key triggers for dendritic cell (DC) activation and subsequent T cell priming. Moreover, we explore the contributions of non-apoptotic RCD pathways, including necroptosis, ferroptosis, and pyroptosis, to tumor immunity within the TME. Emerging evidence suggests that these alternative cell death modalities possess immunogenic properties and can synergize with conventional treatments to bolster anti-tumor immune responses. Furthermore, we discuss the therapeutic implications of targeting the TME for cancer treatment, highlighting strategies to harness immunogenic cell death and manipulate non-apoptotic cell death pathways for therapeutic benefit. By elucidating the intricate crosstalk between cancer cell death and immune modulation within the TME, this review aims to pave the way for the development of novel cancer therapies that exploit the interplay between cell death mechanisms and tumor immunity and overcome Challenges in the Development and implementation of Novel Therapies.

Duplication and neofunctionalization of a horizontally transferred xyloglucanase as a facet of the Red Queen coevolutionary dynamic

Oomycete protists share phenotypic similarities with fungi, including the ability to cause plant diseases, but branch in a distant region of the tree of life. It has been suggested that multiple horizontal gene transfers (HGTs) from fungi-to-oomycetes contributed to the evolution of plant-pathogenic traits. These HGTs are predicted to include secreted proteins that degrade plant cell walls, a barrier to pathogen invasion and a rich source of carbohydrates. Using a combination of phylogenomics and functional assays, we investigate the diversification of a horizontally transferred xyloglucanase gene family in the model oomycete species Phytophthora sojae. Our analyses detect 11 xyloglucanase paralogs retained in P. sojae. Using heterologous expression in yeast, we show consistent evidence that eight of these paralogs have xyloglucanase function, including variants with distinct protein characteristics, such as a long-disordered C-terminal extension that can increase xyloglucanase activity. The functional variants analyzed subtend a phylogenetic node close to the fungi-to-oomycete transfer, suggesting the horizontally transferred gene was a bona fide xyloglucanase. Expression of three xyloglucanase paralogs in Nicotiana benthamiana triggers high-reactive oxygen species (ROS) generation, while others inhibit ROS responses to bacterial immunogens, demonstrating that the paralogs differentially stimulate pattern-triggered immunity. Mass spectrometry of detectable enzymatic products demonstrates that some paralogs catalyze the production of variant breakdown profiles, suggesting that secretion of variant xyloglucanases increases efficiency of xyloglucan breakdown as well as diversifying the damage-associated molecular patterns released. We suggest that this pattern of neofunctionalization and the variant host responses represent an aspect of the Red Queen host-pathogen coevolutionary dynamic.

In vitro cell killing proficiency of autologous immune cells educated with allogenic multi-cancer tumor cell lysate.

e14675 Background: Allogenic tumor lysate therapies offer a novel approach in oncology, to initiate a measured immune system response to combat cancer. Loaded with tumor antigens, these lysates stimulate the patient's immune system for targeted cancer cell destruction. In vitro data suggesting efficacy is pivotal prior to clinical trials. Per-C-Vax is a novel, organ-agnostic, allogenic tumor lysate immuno-stimulant, and is intended to target solid organ malignancies via subcutaneous injection. It is derived from a multi-organ cell-line cocktail, which has been induced into apoptosis to capture diverse Tumor Associated Molecular Patterns (TAMPs) and Damage Associated Molecular Patterns (DAMPs). Our study assesses Per-C-Vax-educated patient-specific immunocytes' in-vitro cell-killing activity against their autologous tumor cells. Methods: We collected simultaneous blood and tumor tissue samples from 22 patients with solid organ tumors under approved protocols. The cohort included 10 head and neck (45%), 4 breast (18%), and 8 other cancers (36%) comprising colorectal, ovary, lung, gallbladder, prostate, uterine, cervix, and gastric cancers. Peripheral Blood Mononuclear Cells (PBMCs) from a 10 ml venous draw were split into control and Per-C-Vax incubated aliquots for each patient. Approximately 1,000 tumor cells, stained with Calcein AM, were co-cultured with about 20,000 cells from each of the Per-C-Vax and control PBMC aliquots (autologous). Imaging-based pre- and post-co-culture counts determined the cell-kill rate for each aliquot. Results: PBMCs not treated with Per C Vax showed a significantly reduced cancer cell killing activity against autologous tumor cells compared to Per-C-Vax -treated PBMCs, suggesting the tolerogenic characteristic of immunocytes to the malignancy (0%-32%, 95% CI 10.16 to 16.65). On the other hand, PBMCs treated with Per-C-Vax showed significant cell-kill activity ranging from 46% to 84% in head and neck (mean 62.1%, 95% CI 52.67 to 71.53), 51% to 73% in breast (mean 62%, 95% CI 44.33 to 79.67), 38% to 87% in other cancers (colorectal, ovary, lung, gallbladder, prostate, uterine, cervix, gastric) (mean 50.87%, 95% CI 37.71 to 64.04). A comparative analysis between the untreated group and the group treated with Per-C-Vax revealed a statistically significant result with a p-value <0.005. Conclusions: Patient-specific PBMCs incubated with Per-C-Vax tumor lysate immuno-stimulant show significant autologous cancer cell killing activity against multiple cancers in vitro. Human trials to establish safety and efficacy are planned.

The role of the cGAS-STING signaling pathway in viral infections, inflammatory and autoimmune diseases.

Pattern recognition receptors are an essential part of the immune system, which detect pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) and help shape both innate and adaptive immune responses. When dsDNA is present, cyclic GMP-AMP Synthase (cGAS) produces a second messenger called cyclic GMP-AMP (cGAMP), which then triggers an adaptor protein called STING, and eventually activates the expression of type I interferon (IFN) and pro-inflammatory cytokines in immune cells. The cGAS-STING signaling pathway has been receiving a lot of attention lately as a key immune-surveillance mediator. In this review, we summarize the present circumstances of the cGAS-STING signaling pathway in viral infections and inflammatory diseases, as well as autoimmune diseases. Modulation of the cGAS-STING signaling pathway provides potential strategies for treating viral infections, inflammatory diseases, and autoimmune diseases.

Type-I interferon shapes peritoneal immunity in cirrhosis and drives caspase-5-mediated progranulin release upon infection

Gut bacterial translocation contributes to immune dysfunction and spontaneous bacterial peritonitis (SBP) in cirrhosis. We hypothesized that exposure of peritoneal macrophages (PMs) to bacterial DNA results in type-I interferon (IFN) production, shaping subsequent immune responses, inflammasome activation, and the release of damage-associated molecular patterns (DAMPs). PMs from patients with cirrhosis were stimulated with E.coli single-stranded DNA (ssDNA), lipopolysaccharide and IFN, or infected with E.coli, S.aureus, and Group B streptococcus invitro. Cytokine release, inflammasome activation, and DAMP release were quantified by quantitative-PCR, ELISA, western blots, and reporter cells employing primary PMs, monocytes, and caspase-deficient THP-1 macrophages. Serum progranulin concentration was correlated with transplant-free survival in 77 patients with SBP. E.coli ssDNA induced strong type-I IFN activity in PMs and monocytes, priming them for enhanced lipopolysaccharide-mediated tumor necrosis factor production without inducing toll-like receptor 4 tolerance. During invitro macrophage bacterial infection, type-I IFN release aligned with upregulated expression of IFN-regulatory factors (IRF)1/2 and guanylate binding proteins (GBP)2/5. PMs upregulated inflammasome-associated proteins and type-I IFN upon E.coli ssDNA exposure and released interleukin-1β upon bacterial infection. Proteomic screening in mouse macrophages revealed progranulin release as being caspase-11-dependent during E.coli infection. PMs and THP-1 macrophages released significant amounts of progranulin when infected with S.aureus or E.coli via gasdermin D in a type-I IFN- and caspase-5-dependent manner. During SBP, PMs upregulated IRF1, GBP2/5 and caspase-5 and higher serum progranulin concentrations were indicative of lower 90-day transplant-free survival after SBP. Type-I IFN shapes peritoneal immune responses and regulates caspase-5-mediated progranulin release during SBP. Patients with cirrhosis exhibit impaired immune responses and increased susceptibility to bacterial infections. This study reveals that type-I interferon responses, triggered by pathogen-associated molecular patterns, are crucial in regulating macrophage activation and priming them for inflammatory responses. Additionally, we elucidate the mechanisms by which type-I interferons promote the release of progranulin from macrophages during spontaneous bacterial peritonitis. Our findings enhance understanding of how bacterial translocation affects immune responses, identify novel biomarkers for inflammasome activation during infections, and point to potential therapeutic targets.

Activation of dendritic cells by a synthesized small molecule for breast cancer vaccination.

e13149 Background: Dendritic cell-based vaccination is a promising immunotherapy for treating various types of cancer. However, tumor-loaded dendritic cells alone have limited treatment efficacy. We have designed a small molecule mimicking the action of damage-associated molecular pattern molecules as an adjuvant for activating dendritic cells in vitro, which demonstrated augmented treatment efficacy. Methods: We aimed at exploring the roles of the synthesized small molecule in augmenting the efficacy of dendritic cell-based cancer vaccination to target triple negative breast cancer. Breast cancer mouse model is constructed using a 4T1 tumor cell line. Data were analyzed using a two-tailed unpaired t-test. Results: Mouse dendritic cells could be activated in vitro by the small molecule as an adjuvant evidenced by increased expression of MHC-I, MHC-II and CD40. Augmented motility of dendritic cells was also observed after treatment with the adjuvant. The signaling of NFkB is remarkably activated in dendritic cells by the adjuvant, which facilitates the maturation of dendritic cells. Engraftment of adjuvant-primed dendritic cells loaded with 4T1 tumor antigen into 4T1 tumor-bearing mice efficiently reduced the tumor size and improved survival, compared with the group treated with dendritic cells only. After injection of adjuvant-activated dendritic cells, both CD4 T cell and CD8 T cell populations were expanded in the spleen and lymph nodes draining the tumor sites. Furthermore, the adjuvant could promote the production of IFN-γ and IL-2, cytokines required for T cell activation and expansion. Next, splenocytes were obtained from individual mice after dendritic cell-based treatment. Upon re-encounter with the tumor antigen in vitro, remarkable expansion of T cell populations with a memory cell feature was confirmed in splenocytes derived from mice that received adjuvant-treated dendritic cells. Consistently, production of TNF-α, IFN-γ and granzyme B, was profoundly increased in these T cells associated with the adjuvant treatment. Conclusions: Our work suggests a novel dendritic cell-based cancer immunotherapy by using a small molecule mimicking the action of damage-associated molecular pattern molecules, which have documented immune potentiation activities.

Crosstalk between Oxidative Stress and Inflammation Induced by Ionizing Radiation in Healthy and Cancerous Cells.

Radiotherapy (RT) is a unique modality in cancer treatment with no replacement in many cases and uses a tumoricidal dose of various ionizing radiation (IR) types to kill cancer cells. It causes oxidative stress through reactive oxygen species (ROS) production or the destruction of antioxidant systems. On the other hand, RT stimulates the immune system both directly and indirectly by releasing danger signals from stress-exposed and dying cells. Oxidative stress and inflammation are two reciprocal and closely related mechanisms, one induced and involved by the other. ROS regulates the intracellular signal transduction pathways, which participate in the activation and expression of pro-inflammatory genes. Reciprocally, inflammatory cells release ROS and immune system mediators during the inflammation process, which drive the induction of oxidative stress. Oxidative stress or inflammation-induced damages can result in cell death (CD) or survival mechanisms that may be destructive for normal cells or beneficial for cancerous cells. The present study has focused on the radioprotection of those agents with binary effects of antioxidant and anti-inflammatory mechanisms IR-induced CD.

Circulating metabolic profiling as a biomarker for immune checkpoint blockade efficacy.

2564 Background: Immune checkpoint blockade (ICB) has changed the treatment landscape of non-small cell lung cancer (NSCLC). Despite being the mainstay of treatment for advanced NSCLC, the development of resistance to ICB is common. Hence, identifying biomarkers that can be used to select patients (pts) who will benefit from ICB is crucial. Here, we interrogate the circulating metabolome to identify the metabolic parameters associated with ICB effectiveness. Methods: This single-center retrospective analysis used a mass-spectrometry (MS) targeted metabolomics approach to assess the relative abundance of 115 metabolites in baseline (pre-ICB) plasma of 55 pts with advanced NSCLC. Pts treated with ICB at the Princess Margaret Cancer Centre from 2018-2020 were included. Electronic medical records were reviewed to collect clinicopathological and treatment data. All pts had tumour next-generation sequencing (NGS) by a targeted gene panel. Descriptive statistics were used to summarize the patient characteristics, treatment modalities, and outcomes. Time-to-event outcomes were analyzed using the Kaplan-Meier method. Progression-free survival (rwPFS) and overall survival (rwOS) were defined as the time from the first treatment dose to radiographic or clinical progression or death from any cause and time from the first dose of treatment to death from any cause, respectively. The response rate was assessed using RECIST 1.1. Statistical significance was determined as a p-value <0.05. Results: Amongst the 55 pts profiled, 42 (76.3%) had adenocarcinoma. The most common molecular alterations included KRAS (n=15), BRAF non-V600 (n=5), and METex14 (n=3). The median PD-L1 score was 65% (IQR 22.5 – 59%). All pts were treated with a single-agent PD-1 inhibitor, and 67.2% of pts received ICB as the first line of treatment. Metabolomic analysis of the pre-ICB initiation plasma samples identified 8 metabolites whose relative abundance significantly differed between responding (CR/PR) and non-responding pts (SD/PD). Amongst these metabolites was the endogenous danger signal Glucosylceramide (d 18:1/24:0), whose abundance was increased in the plasma of the responding pts. When we stratified pts by circulating levels of glucosylceramide, we found a statistically significant higher rwPFS (8.5 vs. 1.6 months, p=0.0001) and rwOS (13.5 vs. 6.1 months, p=0.0001) in pts who had high baseline plasma levels of Glucosylceramide (d 18:1/24:0) (top 50%) versus those with lower levels (bottom 50%). Conclusions: Utilizing a targeted metabolomics approach, we identified the metabolite and endogenous danger signal glucosylceramide (d 18:1/24:0) as a potential metabolic biomarker of response to ICB therapy. Future work will aim to validate this finding in a larger cohort and to understand the biological mechanism underpinning this correlation.

Nanoparticle-mediated celastrol ER targeting delivery amplify immunogenic cell death in melanoma

IntroductionChemoimmunotherapy, which benefits from the combination of chemotherapy and immunotherapy, has emerged as a promising strategy in cancer treatment. However, effectively inducing a robust immune response remains challenging due to the limited responsiveness across patients. Endoplasmic reticulum (ER) stress is essential for activating intracellular signaling pathways associated with immunogenic cell death (ICD), targeting drugs to ER might enhance ER stress and improve ICD-related immunotherapy. ObjectivesTo improve the immune response of Chemoimmunotherapy. MethodsER targeting nanoparticles TSE-CEL/NP were constructed to enhance immunogenic cancer cell death. Flow cytometry, confocal microscope, TEM and immunofluorescence were used to evaluate the ER targeting effect and immunogenic tumor cell death in vitro on B16F10 tumor cells. Unilateral and bilateral tumor models were constructed to investigate the efficacy of anti-tumor and immunotherapy in vivo. Lung metastasis B16F10 melanoma tumor-bearing mice were used to assess the anti-metastasis efficacy. ResultsTSE-CEL/NP could specially accumulate in ER, thereby induce ER stress. High ER stress trigger the exposure of CRT, the extracellular release of HMGB1 and ATP. These danger signals subsequently promote the recruitment and maturation of dendritic cells (DCs), which in turn increase the proliferation of cytotoxic T lymphocytes (CD8+ T cells), ultimately resulted in an improved immunotherapy efficacy against melanoma. Invivo experiments showed that TSE-CEL/NP exhibits excellent antitumor efficacy and triggers a strong immune response. ConclusionOur findings demonstrated that celastrol ER targeting delivery could amplify immunogenic cell death in melanoma, which provide experimental basis for melanoma immunotherapy.

Lipophilic fluorescent products as a potential biomarker of oxidative stress: A link between central (brain) and peripheral (blood).

Oxidative stress plays a key role in brain damage because of the sensitivity of brain tissue to oxidative damage. Biomarkers with easy measurement can be a candidate for reflecting the oxidative stress issue in humans. For this reason, we need to focus on specific metabolic products of the brain. End products of free radical reactions such as malondialdehydes form fluorescent products known as lipophilic fluorescent products (LFPs). The distinctive feature of LFPs is their autofluorescent properties. LFPs are detectable in the brain and cerebrospinal fluid. Furthermore, because of the diffusion into the bloodstream, these lipophilic molecules can be detected in the blood. Accumulations of these compounds produce more reactive oxygen species and increase the sensitivity of cells to oxidative damage. Hence, LFPs can be considered a danger signal for neurons and can be introduced as a strong index of oxidative damage both in the central and in the peripheral.