Mechanisms Of Crosstalk Research Articles

Crosstalking with Dendritic Cells: A Path to Engineer Advanced T Cell Immunotherapy.

Crosstalk between dendritic cells (DCs) and T cells plays a crucial role in modulating immune responses in natural and pathological conditions. DC-T cell crosstalk is achieved through contact-dependent (i.e., immunological synapse) and contact-independent mechanisms (i.e., cytokines). Activated DCs upregulate co-stimulatory signals and secrete proinflammatory cytokines to orchestrate T cell activation and differentiation. Conversely, activated T helper cells "license" DCs towards maturation, while regulatory T cells (Tregs) silence DCs to elicit tolerogenic immunity. Strategies to efficiently modulate the DC-T cell crosstalk can be harnessed to promote immune activation for cancer immunotherapy or immune tolerance for the treatment of autoimmune diseases. Here, we review the natural crosstalk mechanisms between DC and T cells. We highlight bioengineering approaches to modulate DC-T cell crosstalk, including conventional vaccines, synthetic vaccines, and DC-mimics, and key seminal studies leveraging these approaches to steer immune response for the treatment of cancer and autoimmune diseases.

Redox signalling in plant-nematode interactions: Insights into molecular crosstalk and defense mechanisms.

Plant-parasitic nematodes, specifically cyst nematodes (CNs) and root-knot nematodes (RKNs), pose significant threats to global agriculture, leading to substantial crop losses. Both CNs and RKNs induce permanent feeding sites in the root of their host plants, which then serve as their only source of nutrients throughout their lifecycle. Plants deploy reactive oxygen species (ROS) as a primary defense mechanism against nematode invasion. Notably, both CNs and RKNs have evolved sophisticated strategies to manipulate the host's redox environment to their advantage, with each employing distinct tactics to combat ROS. In this review, we have focused on the role of ROS and its scavenging network in interactions between host plants and CNs and RKNs. Overall, this review emphasizes the complex interplay between plant defense mechanism, redox signallingand nematode survival tactics, suggesting potential avenues for developing innovative nematode management strategies in agriculture.

Molecular crosstalk and putative mechanisms underlying mitochondrial quality control: The hidden link with methylmercury-induced cognitive impairment

Methylmercury (MeHg) is a neurotoxin associated with foetal neurodevelopmental and adult cognitive deficits. Neurons are highly dependent on the tricarboxylic acid cycle and oxidative phosphorylation to produce ATP and meet their high energy demands. Therefore, mitochondrial quality control (MQC) is critical for neuronal homeostasis. While existing studies have generated a wealth of data on the toxicity of MeHg, the complex cascades and molecular pathways governing the mitochondrial network remain to be elucidated. Here, 0.6, 1.2 and 2.4 mg/kg body weight of MeHg were administered intragastrically to pregnant Sprague Dawley rats to model maternal MeHg exposure. The results of the in vivo study revealed that MeHg-treated rats tended to perform more directionless repetitive strategies in the Morris Water Maze and fewer target-orientation strategies than control offspring. Moreover, pathological injury and synaptic toxicity were observed in the hippocampus. Transmission electron microscopy (TEM) demonstrated that the autophagosomes encapsulated damaged mitochondria, while showing a typical mitochondrial fission phenotype, which was supported by the activation of PINK1-dependent key regulators of mitophagy. Moreover, there was upregulation of DRP1 and FIS1. Additionally, MeHg compensation promoted mitochondrial biogenesis, as evidenced by the activation of the mitochondrial PGC1-α-NRF1-TFAM signalling pathway. Notably, SIRT3/AMPK was activated by MeHg, and the expression and activity of p-AMPK, p-LKB1 and SIRT3 were consistently coordinated. Collectively, these findings provide new insights into the potential molecular mechanisms regulating MeHg-induced cognitive deficits through SIRT3/AMPK MQC network coordination.

A2AR-mediated CXCL5 upregulation on macrophages promotes NSCLC progression via NETosis

Tumor-associated macrophages (TAMs) are abundant in tumors and interact with tumor cells, leading to the formation of an immunosuppressive microenvironment and tumor progression. Although many studies have explored the mechanisms underlying TAM polarization and its immunosuppressive functions, understanding of its progression remains limited. TAMs promote tumor progression by secreting cytokines, which subsequently recruit immunosuppressive cells to suppress the antitumor immunity. In this study, we established an in vitro model of macrophage and non-small cell lung cancer (NSCLC) cell co-culture to explore the mechanisms of cell-cell crosstalk. We observed that in NSCLC, the C-X-C motif chemokine ligand 5 (CXCL5) was upregulated in macrophages because of the stimulation of A2AR by adenosine. Adenosine was catalyzed by CD39 and CD73 in macrophages and tumor cells, respectively. Nuclear factor kappa B (NFκB) mediated the A2AR stimulation of CXCL5 upregulation in macrophages. Additionally, CXCL5 stimulated NETosis in neutrophils. Neutrophil extracellular traps (NETs)-treated CD8+ T cells exhibited upregulation of exhaustion-related and cytosolic DNA sensing pathways and downregulation of effector-related genes. However, A2AR inhibition significantly downregulated CXCL5 expression and reduced neutrophil infiltration, consequently alleviating CD8+ T cell dysfunction. Our findings suggest a complex interaction between tumor and immune cells and its potential as therapeutic target.

Cross-talk between BCKDK-mediated phosphorylation and STUB1-dependent ubiquitination degradation of BCAT1 promotes GBM progression

Branched-chain amino acid transferase 1 (BCAT1) is highly expressed in multiple cancers and is associated with poor prognosis, particularly in glioblastoma (GBM). However, the post-translational modification (PTM) mechanism of BCAT1 is unknown. Here, we investigated the cross-talk mechanisms between phosphorylation and ubiquitination modifications in regulating BCAT1 activity and stability. We found that BCAT1 is phosphorylated by branched chain ketoacid dehydrogenase kinase (BCKDK) at S5, S9, and T312, which increases its catalytic and antioxidant activity and stability. STUB1 (STIP1 homology U-box-containing protein 1), the first we found and reported E3 ubiquitin ligase of BCAT1, can also be phosphorylated by BCKDK at the S19 site, which disrupts the interaction with BCAT1 and inhibits its degradation. In addition, we demonstrate through in vivo and in vitro experiments that BCAT1 phosphorylation inhibiting its ubiquitination at multiple sites is associated with GBM proliferation and that inhibition of the BCKDK-BCAT1 axis enhances the sensitivity to temozolomide (TMZ). Overall, we identified novel mechanisms for the regulation of BCAT1 modification and elucidated the importance of the BCKDK-STUB1-BCAT1 axis in GBM progression.

Crosstalk interactions between transcription factors ERRα and PPARα assist PPARα-mediated gene expression

ObjectiveThe peroxisome proliferator-activated receptor α (PPARα) is a transcription factor driving target genes involved in fatty acid β-oxidation. To what extent various PPARα interacting proteins may assist its function as a transcription factor is incompletely understood. An ORFeome-wide unbiased mammalian protein–protein interaction trap (MAPPIT) using PPARα as bait revealed a PPARα-ligand-dependent interaction with the orphan nuclear receptor estrogen-related receptor α (ERRα). The goal of this study was to characterize the nature of the interaction in depth and to explore whether it was of physiological relevance. MethodsWe used orthogonal protein–protein interaction assays and pharmacological inhibitors of ERRα in various systems to confirm a functional interaction and study the impact of crosstalk mechanisms. To characterize the interaction surfaces and contact points we applied a random mutagenesis screen and structural overlays. We pinpointed the extent of reciprocal ligand effects of both nuclear receptors via coregulator peptide recruitment assays. On PPARα targets revealed from a genome-wide transcriptome analysis, we performed an ERRα chromatin immunoprecipitation analysis on both fast and fed mouse livers. ResultsRandom mutagenesis scanning of PPARα's ligand-binding domain and coregulator profiling experiments supported the involvement of (a) bridging coregulator(s), while recapitulation of the interaction in vitro indicated the possibility of a trimeric interaction with RXRα. The PPARα·ERRα interaction depends on 3 C-terminal residues within helix 12 of ERRα and is strengthened by both PGC1α and serum deprivation. Pharmacological inhibition of ERRα decreased the interaction of ERRα to ligand-activated PPARα and revealed a transcriptome in line with enhanced mRNA expression of prototypical PPARα target genes, suggesting a role for ERRα as a transcriptional repressor. Strikingly, on other PPARα targets, including the isolated PDK4 enhancer, ERRα behaved oppositely. Chromatin immunoprecipitation analyses demonstrate a PPARα ligand-dependent ERRα recruitment onto chromatin at PPARα-binding regions, which is lost following ERRα inhibition in fed mouse livers. ConclusionsOur data support the coexistence of multiple layers of transcriptional crosstalk mechanisms between PPARα and ERRα, which may serve to finetune the activity of PPARα as a nutrient-sensing transcription factor.

STING agonist diABZI enhances the cytotoxicity of T cell towards cancer cells

Antigen-specific T cell receptor-engineered T cell (TCR-T) based immunotherapy has proven to be an effective method to combat cancer. In recent years, cross-talk between the innate and adaptive immune systems may be requisite to optimize sustained antigen-specific immunity, and the stimulator of interferon genes (STING) is a promising therapeutic target for cancer immunotherapy. The level of expression or presentation of antigen in tumor cells affects the recognition and killing of tumor cells by TCR-T. This study aimed at investigating the potential of innate immune stimulation of T cells and engineered T cells to enhance immunotherapy for low-expression antigen cancer cells. We systematically investigated the function and mechanism of cross-talk between STING agonist diABZI and adaptive immune systems. We established NY-ESO-1 full knockout Mel526 cells for this research and found that diABZI activated STING media and TCR signaling pathways. In addition, the results of flow cytometry showed that antigens presentation from cancer cells induced by STING agonist diABZI also improved the affinity of TCR-T cells function against tumor cells in vitro and in vivo. Our findings revealed that diABZI enhanced the immunotherapy efficacy of TCR-T by activating STING media and TCR signaling pathways, improving interferon-γ expression, and increasing antigens presentation of tumor cells. This indicates that STING agonist could be used as a strategy to promote TCR-T cancer immunotherapy.

Microbial perspective on the skin-gut axis and atopic dermatitis.

Atopic dermatitis (AD) is a relapsing inflammatory skin condition that has become a global health issue with complex etiology and mounting prevalence. The association of AD with skin and gut microbiota has been revealed by virtue of the continuous development of sequencing technology and genomics analysis. Also, the gut-brain-skin axis and its mutual crosstalk mechanisms have been gradually verified. Accordingly, the microbiota-skin-gut axis also plays an important role in allergic skin inflammation. Herein, we reviewed the relationship between the microbiota-skin-gut axis and AD, explored the underlying signaling molecules and potential pathways, and focused on the potential mechanisms of probiotics, antimicrobial peptides (AMPs), coagulase-negative staphylococci transplantation, fecal microbiota transplantation, AMPs, and addition of essential fatty acids in alleviating AD, with the aim to provide a new perspective for targeting microbiota in the treatment of allergic skin inflammation.

Reactive oxygen species (ROS) modulate nitrogen signaling using temporal transcriptome analysis in foxtail millet.

Reactive oxygen species (ROS) is a chemically reactive chemical substance containing oxygen and a natural by-product of normal oxygen metabolism. Excessive ROS affect the growth process of crops, which will lead to the decrease of yield. Nitrogen, as a critical nutrient element in plants and plays a vital role in plant growth and crop production. Nitrate is the primary nitrogen source available to plants in agricultural soil and various natural environments. However, the molecular mechanism of ROS-nitrate crosstalk is still unclear. In this study, we used the foxtail millet (Setaria italica L.) as the material to figure it out. Here, we show that excessive NaCl inhibits nitrate-promoted plant growth and nitrogen use efficiency (NUE). NaCl induces ROS accumulation in roots, and ROS inhibits nitrate-induced gene expression in a short time. Surprisingly, low concentration ROS slight promotes and high concentration of ROS inhibits foxtail millet growth under long-term H2O2 treatment. These results may open a new perspective for further exploration of ROS-nitrate signaling pathway in plants.

The molecular mechanism of macrophage-adipocyte crosstalk in maintaining energy homeostasis.

Interactions between macrophages and adipocytes in adipose tissue are critical for the regulation of energy metabolism and obesity. Macrophage polarization induced by cold or other stimulations can drive metabolic reprogramming of adipocytes, browning, and thermogenesis. Accordingly, investigating the roles of macrophages and adipocytes in the maintenance of energy homeostasis is critical for the development of novel therapeutic approaches specifically targeting macrophages in metabolic disorders such as obesity. Current review outlines macrophage polarization not only regulates the release of central nervous system and inflammatory factors, but controls mitochondrial function, and other factor that induce metabolic reprogramming of adipocytes and maintain energy homeostasis. We also emphasized on how the adipocytes conversely motivate the polarization of macrophage. Exploring the interactions between adipocytes and macrophages may provide new therapeutic strategies for the management of obesity-related metabolic diseases.

Aged-vascular niche hinders osteogenesis of mesenchymal stem cells through paracrine repression of Wnt-axis.

Age-induced decline in osteogenic potential of bone marrow mesenchymal stem cells (BMSCs) potentiates osteoporosis and increases the risk for bone fractures. Despite epidemiology studies reporting concurrent development of vascular and bone diseases in the elderly, the underlying mechanisms for the vascular-bone cross-talk in aging are largely unknown. In this study, we show that accelerated endothelial aging deteriorates bone tissue through paracrine repression of Wnt-driven-axis in BMSCs. Here, we utilize physiologically aged mice in conjunction with our transgenic endothelial progeria mouse model (Hutchinson-Gilford progeria syndrome; HGPS) that displays hallmarks of an aged bone marrow vascular niche. We find bone defects associated with diminished BMSC osteogenic differentiation that implicate the existence of angiocrine factors with long-term inhibitory effects. microRNA-transcriptomics of HGPS patient plasma combined with aged-vascular niche analyses in progeria mice reveal abundant secretion of Wnt-repressive microRNA-31-5p. Moreover, we show that inhibition of microRNA-31-5p as well as selective Wnt-activator CHIR99021 boosts the osteogenic potential of BMSCs through de-repression and activation of the Wnt-signaling, respectively. Our results demonstrate that the vascular niche significantly contributes to osteogenesis defects in aging and pave the ground for microRNA-based therapies of bone loss in elderly.

NFĸB signaling drives myocardial injury via CCR2+ macrophages in a preclinical model of arrhythmogenic cardiomyopathy.

Nuclear factor κ-B (NFκB) is activated in iPSC-cardiac myocytes from patients with arrhythmogenic cardiomyopathy (ACM) under basal conditions, and inhibition of NFκB signaling prevents disease in Dsg2mut/mut mice, a robust mouse model of ACM. Here, we used genetic approaches and single-cell RNA-Seq to define the contributions of immune signaling in cardiac myocytes and macrophages in the natural progression of ACM using Dsg2mut/mut mice. We found that NFκB signaling in cardiac myocytes drives myocardial injury, contractile dysfunction, and arrhythmias in Dsg2mut/mut mice. NFκB signaling in cardiac myocytes mobilizes macrophages expressing C-C motif chemokine receptor-2 (CCR2+ cells) to affected areas within the heart, where they mediate myocardial injury and arrhythmias. Contractile dysfunction in Dsg2mut/mut mice is caused both by loss of heart muscle and negative inotropic effects of inflammation in viable muscle. Single nucleus RNA-Seq and cellular indexing of transcriptomes and epitomes (CITE-Seq) studies revealed marked proinflammatory changes in gene expression and the cellular landscape in hearts of Dsg2mut/mut mice involving cardiac myocytes, fibroblasts, and CCR2+ macrophages. Changes in gene expression in cardiac myocytes and fibroblasts in Dsg2mut/mut mice were dependent on CCR2+ macrophage recruitment to the heart. These results highlight complex mechanisms of immune injury and regulatory crosstalk between cardiac myocytes, inflammatory cells, and fibroblasts in the pathogenesis of ACM.

Thermal flux manipulation on the silicon photonic chip to suppress the thermal crosstalk

The integration density of silicon photonic integrated circuit (PIC) is ultimately constrained by various crosstalk mechanisms on the chip. Among them, the most prominent limiting factor is the thermal crosstalk due to the wide use of the thermo-optic effect. High-density silicon PICs strongly demand an advanced structure with better thermal crosstalk suppression ability than the traditional air isolation trench. Inspired by the thermal-metamaterial based on the scattering-cancellation method, we demonstrate a closed heat shield (CHS) structure on a silicon PIC chip, which can manipulate the thermal flux to bypass the temperature-sensitive silicon photonics components. The on-chip CHS structure is a bilayer cylindrical shell fabricated by the standard silicon photonics processing flow. Its outer and inner shell layers are formed by a 6-μm-wide interconnection metal and 4-μm-wide air trench, respectively. Plenty of temperature-sensitive micro-ring resonators inside the CHS are used to probe the temperature profile. The measurement results show that the CHS can reduce the local temperatures by 50%/44%/36% at the locations 29/41/83 μm away from the external heater. In contrast, the conventional air trench of the same dimension reduces the local temperatures by 32%/28%/21% at the same positions. In addition, the response time of the thermal field inside the CHS is around one-half of that in the conventional air trench. Furthermore, the simulation result indicates that if the outer shell of the CHS can contact with the silicon substrate by utilizing the through-silicon-via structure, the thermal crosstalk suppression ability can be improved significantly.

Crosstalk of lysyl oxidase-like 1 and lysyl oxidase prolongs their half-lives and regulates liver fibrosis through Notch signal.

Lysyl oxidase (LOX) family members (LOX and LOXL1 to 4) are crucial copper-dependent enzymes responsible for cross-linking collagen and elastin. Previous studies have revealed that LOX and LOXL1 are the most dramatically dysregulated LOX isoforms during liver fibrosis. However, the crosstalk between them and the underlying mechanisms involved in the profibrotic behaviors of HSCs, as well as the progression of liver fibrosis, remain unclear. pCol9GFP-HS4,5Tg mice, Loxl1fl/flGfapCre mice, human HSC line, and primary HSCs were enrolled to study the dysregulation pattern, profibrotic roles, and the potential mechanisms of LOX and LOXL1 interaction involved in the myofibroblast-like transition of HSCs and liver fibrogenesis. LOX and LOXL1 were synergistically upregulated during liver fibrogenesis, irrespective of etiology, together orchestrating the profibrotic behaviors of HSCs. LOX and LOXL1 coregulated in HSCs, whereas LOXL1 dominated in the coregulation loop. Interestingly, the interaction between LOXL1 and LOX prolonged their half-lives, specifically enhancing the Notch signal-mediated myofibroblast-like transition of HSCs. Selective disruption of Loxl1 in Gfap+ HSCs deactivated the Notch signal, inhibited HSC activation, and relieved carbon tetrachloride-induced liver fibrosis. Our current study confirmed the synergistic roles and the underlying mechanisms of LOXL1 and LOX crosstalk in the profibrotic behaviors of HSCs and liver fibrosis progression, providing experimental evidence for further clear mechanism-based anti-LOXL1 strategy development in the therapy of liver fibrosis.

Identifying MS4A6A+ macrophages as potential contributors to the pathogenesis of nonalcoholic fatty liver disease, periodontitis, and type 2 diabetes mellitus

PurposeConcrete epidemiological evidence has suggested the mutually-contributing effect respectively between nonalcoholic fatty liver disease (NAFLD), type 2 diabetes mellitus (T2DM), and periodontitis (PD); however, their shared crosstalk mechanism remains an open issue. MethodThe NAFLD, PD, and T2DM-related datasets were obtained from the NCBI GEO repository. Their common differentially expressed genes (DEGs) were identified and the functional enrichment analysis performed by the DAVID platform determined relevant biological processes and pathways. Then, the STRING database established a PPI network of such DEGs and topological analysis through Cytoscape 3.7.1 software along with the machine-learning analysis by the least absolute shrinkage and selection operator (LASSO) algorithm screened out hub characteristic genes. Their efficacy was validated by external datasets using the receiver operating characteristic (ROC) curve, and gene expression and location of the most robust one was determined using single-cell sequencing and immunohistochemical staining. Finally, the promising drugs were predicted through the CTD database, and the CB-DOCK 2 and Pymol platform mimicked molecular docking. ResultIntersection of differentially expressed genes from three datasets identified 25 shared DEGs of the three diseases, which were enriched in MHC II-mediated antigen presenting process. PPI network and LASSO machine-learning analysis determined 4 feature genes, of which the MS4A6A gene mainly expressed by macrophages was the hub gene and key immune cell type. Molecular docking simulation chosen fenretinide as the most promising medicant for MS4A6A+ macrophages. ConclusionMS4A6A+ macrophages were suggested to be important immune-related mediators in the progression of NAFLD, PD, and T2DM pathologies.

P‐9.13: Research on Horizontal Crosstalk Mechanism of LCD Display with Demultiplexer

P‐9.13: Research on Horizontal Crosstalk Mechanism of LCD Display with Demultiplexer

A multi-omics investigation of the lung injury induced by PM2.5 at environmental levels via the lung-gut axis

Long-term exposure to fine particulate matter (PM2.5) posed injury for gastrointestinal and respiratory systems, ascribing with the lung-gut axis. However, the cross-talk mechanisms remain unclear. Here, we attempted to establish the response networks of lung-gut axis in mice exposed to PM2.5 at environmental levels. Male Balb/c mice were exposed to PM2.5 (dose of 0.1, 0.5, and 1.0 mg/kg) collected from Chengdu, China for 10 weeks, through intratracheally instillation, and examined the effect of PM2.5 on lung functions of mice. The changes of lung and gut microbiota and metabolic profiles of mice in different groups were determined. Furthermore, the results of multi-omics were conjointly analyzed to elucidate the primary microbes and the associated metabolites in lung and gut responsible for PM2.5 exposure. Accordingly, the cross-talk network and key pathways between lung-gut axis were established. The results indicated that exposed to PM2.5 0.1 mg/kg induced obvious inflammations in mice lung, while emphysema was observed at 1.0 mg/kg. The levels of metabolites guanosine, hypoxanthine, and hepoxilin B3 increased in the lung might contribute to lung inflammations in exposure groups. For microbiotas in lung, PM2.5 exposure significantly declined the proportions of Halomonas and Lactobacillus. Meanwhile, the metabolites in gut including L-tryptophan, serotonin, and spermidine were up-regulated in exposure groups, which were linked to the decreasing of Oscillospira and Helicobacter in gut. Via lung-gut axis, the activations of pathways including Tryptophan metabolism, ABC transporters, Serotonergic synapse, and Linoleic acid metabolism contributed to the cross-talk between lung and gut tissues of mice mediated by PM2.5. In summary, the microbes including Lactobacillus, Oscillospira, and Parabacteroides, and metabolites including hepoxilin B3, guanosine, hypoxanthine, L-tryptophan, and spermidine were the main drivers. In this lung-gut axis study, we elucidated some pro- and pre-biotics in lung and gut microenvironments contributed to the adverse effects on lung functions induced by PM2.5 exposure.

Abstract 5767: DNA binding mechanism of prostate cancer target ONECUT2

Abstract Transcription factors (TF) are essential determinants of gene regulation and their aberrant expression frequently drive cancers. The binding of TFs to cognate DNA is central to their role in gene regulation. An understanding of their DNA binding mechanism is, therefore, of fundamental and therapeutic relevance. Generally, TFs bind DNA through defined DNA binding domains. The CUT and homeodomain are two such evolutionarily conserved elements found paired in multiple transcription factor families including ONECUT (OC), POU, CUX and SATB. However, the mechanism of cross-talk between the CUT and homeodomain to coordinatedly bind DNA remains unknown. Here, we report an integrative DNA binding analyses of the transcription factor OC2, a promising target for treatment of aggressive prostate cancer. We demonstrate that the homeodomain of OC2 thermodynamically stabilizes the DNA binding through allosteric modulation of CUT. The binding energetics is further dependent on base interactions by evolutionarily conserved amino acids in both CUT and homeodomain. In addition, we have discovered a unique arginine pair in the homeodomain, conserved within the ONECUT family, but not across POU and SATB families. We show that one arginine of this pair binds distinctly to DNA in respective complexes formed by OC2 and OC1. Notably, the above base-interactions are crucial for OC2-driven disease progression in a prostate cancer cell-line model. Taken together, our studies show that base-interactions by the evolutionarily conserved residues determine the basic DNA binding functionality through establishing favorable thermodynamics, while interactions by the arginine motif impart family-specific attributes. These findings also establish the homeodomain as a key regulator of DNA binding by OC2 and provide a mechanism for cooperative DNA binding by the CUT-homeodomain module in general. Importantly, these insights reveal vulnerabilities for therapeutic targeting of OC2. Citation Format: Avradip Chatterjee, Brad Gallent, Madhusudhanarao Katiki, Chen Qian, Matthew R. Harter, Michael R. Freeman, Ramachandran Murali. DNA binding mechanism of prostate cancer target ONECUT2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5767.

Abstract 4223: The dual immunestimulatory and immunesuppressive role of MRC5 fibroblast cells in 3D coculture with T cells and tumor cells

Abstract Introduction: Cancer-associated fibroblasts (CAFs) alter anti-tumor immune responses and modulate resistance to immune checkpoint inhibitors. The mechanisms of crosstalk between tumor cells, CAFs, and T cells remain not fully understood, and studying these multicellular interactions requires adoption of novel tools in preclinical research. Here, we established a spheroid co-culture model comprising of tumor cell lines, a fibroblast cell line, and primary human T cells, for studying the immunomodulatory effects of fibroblasts on T cell responses in vitro, in the context of immunotherapies. Methods: For setting up the 3D co-culture, A375 or OV-90 tumor cells were seeded alone or with MRC-5 fibroblasts in 96-well ultra-low attachment round bottom plates in complete growth medium supplemented with 1% Geltrex matrix. The cells were centrifuged, and spheroids were allowed to form for 24 hours. After spheroid initiation, activated T cells and treatments with anti-PD1 antibody or an immune stimulatory drug were added. Spheroid co-cultures were live imaged by Incucyte SX5 for 72 hours taking images every 4 hours, after which treatment responses were evaluated by flow cytometry and measuring IFN-γ accumulation with ELISA. Results: To mimic fibroblast and T cell interactions in tumor microenvironment we built and optimized an in vitro 3D co-culture assay using various tumor cell lines. With this assay we found that depending on the tumor cell interaction, fibroblasts MRC-5 cell line have either immune-suppressive or immune-promoting properties. IFN-γ secretion of T cells in the assay indicated that MRC-5 cells suppressed T cells when co-cultured with A375 cells, while with OV-90 cells T cells activation was enhanced. In addition to the IFN- γ secretion, T cells in MRC-5 OV-90 interaction significantly upregulated 4-1BB expression on both CD4 & CD8 T cells. Interestingly we noticed that MRC-5 fibroblasts could modulate T cell responses to anti-PD-1 treatment in the co-culture depending on the tumor cell used. Conclusion: Here, we established a spheroid co-culture model, which will allow studies of the immunomodulatory crosstalk occurring in tumor microenvironment. Our model revealed a bifunctional role of MRC-5 fibroblasts in altering T cell phenotype. This finding is in line with literature highlighting the ability of fibroblasts to act as either tumor promoters or tumor suppressors depending on tumor microenvironment. We believe that this model, would aid in elucidating mechanisms of interaction between CAFs and T cells, potentially leading to discoveries of novel targets for immuno-oncology drug development. Additional studies, such as gene expression analysis using single cell RNA sequencing, are warranted to further validated the model and to better understand the kinetics of these interaction occurring in the co-culture with various tumor cell lines. Citation Format: Ilona Arnkil, Can Hekim, Elli Narvi, Emmi-Leena Ihantola, Endrit Elbasani, Anil Thotakura. The dual immunestimulatory and immunesuppressive role of MRC5 fibroblast cells in 3D coculture with T cells and tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4223.

Abstract 3988: Deep spatial immunophenotyping of lymphoid aggregates in pancreatic cancer using multi-omic integration of ultra high-plex proteomics and transcriptomics

Abstract The most well-described mechanism of immune evasion in PDAC is exclusion of immune cells by a desmoplastic stroma, but such immune exclusion is not uniform. Through single-nucleus RNA-seq and whole-transcriptome digital spatial profiling of patient tumors, we previously reported marked heterogeneity in the immune composition of PDAC. Here, we developed a novel multi-omics workflow integrating single cell spatial proteomics (96-plex, Akoya Phenocycler®-Fusion) and transcriptomics (990-plex, Nanostring CosMx) in primary patient PDAC tumors to systematically study spatial relationships and functional states of malignant, stromal, and immune cells. Consecutive sections from four independent tumors enabled whole slide proteomic assessment of >3.5M cells, and transcriptomic assessment of >200K cells. Strikingly, we observed an abundance of immune cell aggregates in all tissue sections, up to 57 within a 1 cm2 tumor section. Aggregates were diverse in composition with representation of myeloid cells, CD8+ T cells, and stromal cells, including fibroblasts and nerves. B/T lymphoid aggregates were well vascularized with co-localized CD31+CD34+ endothelial cells and appeared at various phases of maturation towards putative tertiary lymphoid structures (TLSs) containing CD38+ B cells, CD4+ T cells, and CD21+ follicular DCs. These tissues were enriched for interferon stimulated genes in T cells and type 1 interferon response signatures in malignant cells and cancer associated fibroblasts (CAFs) forming inflammatory multicellular hubs that have been associated with improved response to immune checkpoint blockade. Surprisingly, B cells, in addition to macrophages and CD8+ T cells, were major producers of interferon gamma (B cells: 13.7%, macrophages: 36.6%, CD8+ T cells: 12.1% of IFNG+ cells). Finally, to gain insight into the necessary factors for TLS formation in PDAC, we quantified the expression of chemokines and adhesion molecules associated with TLS initiation. Activated dendritic cells expressed CCL19 and CCL21, endothelial cells expressed CCL21, and in concert with inflammatory CAFs (iCAFs), CXCL12. Malignant cells expressed CXCL13 and CCL20 and displayed high levels of cell adhesion molecules ICAM1, VCAM1, and ICAM2. Together, our data suggests that the functions of lymphoid tissue organizer cells can be substituted by endothelial cells, dendritic cells, iCAFs, and, surprisingly, malignant cells. Our spatial immunophenotyping study of pancreatic cancer thus highlights the marked immune heterogeneity of the disease and suggests a central role for malignant and stromal cells in mediating diverse mechanisms of immune evasion and engagement. Functional studies will be conducted to investigate these mechanisms of tumor-immune crosstalk and identify novel immunotherapeutic vulnerabilities for this deadly disease. Citation Format: Dennis Gong, Jingyi Cao, Peter Wang, Niyati Jhaveri, Yue Hou, Bassem B. Cheikh, HaYeun Ji, Mark Gregory, Jason Reeves, Michael Patrick, Carina Shiau, Jennifer Su, Jimmy A. Guo, Joseph M. Beechem, Martin Hemberg, William L. Hwang, Ryan Park. Deep spatial immunophenotyping of lymphoid aggregates in pancreatic cancer using multi-omic integration of ultra high-plex proteomics and transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3988.