Immune Cell Proliferation Research Articles

Thymosin α1 and Its Role in Viral Infectious Diseases: The Mechanism and Clinical Application.

Thymosin α1 (Tα1) is an immunostimulatory peptide that is commonly used as an immune enhancer in viral infectious diseases such as hepatitis B, hepatitis C, and acquired immune deficiency syndrome (AIDS). Tα1 can influence the functions of immune cells, such as T cells, B cells, macrophages, and natural killer cells, by interacting with various Toll-like receptors (TLRs). Generally, Tα1 can bind to TLR3/4/9 and activate downstream IRF3 and NF-κB signal pathways, thus promoting the proliferation and activation of target immune cells. Moreover, TLR2 and TLR7 are also associated with Tα1. TLR2/NF-κB, TLR2/p38MAPK, or TLR7/MyD88 signaling pathways are activated by Tα1 to promote the production of various cytokines, thereby enhancing the innate and adaptive immune responses. At present, there are many reports on the clinical application and pharmacological research of Tα1, but there is no systematic review to analyze its exact clinical efficacy in these viral infectious diseases via its modulation of immune function. This review offers an overview and discussion of the characteristics of Tα1, its immunomodulatory properties, the molecular mechanisms underlying its therapeutic effects, and its clinical applications in antiviral therapy.

DSN enzyme recognition initiated rolling circle amplification (DiRCA) for accurate miRNA detection in immune response and immune repair after trauma

Recent studies have reported that miRNA plays an important role in immune response and immune repair after trauma. By regulating the expression of related target genes, miRNA regulates the production, proliferation, development and immune response of immune cells. Therefore, it is in urgent demand to develop an novel method for miRNA analysis. Rolling circle amplification (RCA), as an attractive isothermal signal amplification strategy, has been widely utilized in constructing miRNA detection assays. However, accurate and sensitive miRNA quantitative determination remains a huge challenge for RCA based approaches. Herein, we propose a DSN enzyme based signal cycle initiated Rolling Circle Amplification assay (DiRCA) for sensitive and accurate miRNA detection. In DiRCA, target miRNA unfolds hairpin structure probe in the detection scaffold, forming a RNA–DNA duplex. DSN enzyme is utilized to specifically digest the DNA sequence in RNA–DNA duplex, releasing miRNA to form a signal cycle; its capability to distinguish one base pair mismatch in RNA–DNA duplex endows DiRCA a high selectivity. Meanwhile, DSN enzyme based cleavage initiates RCA, transcribing G-rich sequences for signal generation. Based on the DSN assisted signal cycle and RCA, DiRCA shows a low limit of detection of 0.43 fM and a superior capability in selectively detecting mismatched miRNA sequences, showing a promising prospect in the early-diagnosis of disease.

Tuning the Biological Activity of PI3Kδ Inhibitor by the Introduction of a Fluorine Atom Using the Computational Workflow.

As a member of the class I PI3K family, phosphoinositide 3-kinase δ (PI3Kδ) is an important signaling biomolecule that controls immune cell differentiation, proliferation, migration, and survival. It also represents a potential and promising therapeutic approach for the management of numerous inflammatory and autoimmune diseases. We designed and assessed the biological activity of new fluorinated analogues of CPL302415, taking into account the therapeutic potential of our selective PI3K inhibitor and fluorine introduction as one of the most frequently used modifications of a lead compound to further improve its biological activity. In this paper, we compare and evaluate the accuracy of our previously described and validated in silico workflow with that of the standard (rigid) molecular docking approach. The findings demonstrated that a properly fitted catalytic (binding) pocket for our chemical cores at the induced-fit docking (IFD) and molecular dynamics (MD) stages, along with QM-derived atomic charges, can be used for activity prediction to better distinguish between active and inactive molecules. Moreover, the standard approach seems to be insufficient to score the halogenated derivatives due to the fixed atomic charges, which do not consider the response and indictive effects caused by fluorine. The proposed computational workflow provides a computational tool for the rational design of novel halogenated drugs.

Identification of a PD1/PD-L1 inhibitor by Structure-Based Pharmacophore Modelling, Virtual Screening, Molecular docking and Biological Evaluation.

PD-1/PD-L1 is a critical druggable target for immunotherapy against sepsis. Chemoinformatics techniques involved the structure-based 3D pharmacophore model development followed by virtual screening of small molecule databases to identify the small molecules against PD-L1 pathway inhibition. Raltitrexed and Safinamide act as potent repurposed drugs, and three other Specs database compounds using in silico methods. These compounds were screened based on the pharmacophore fit score and binding affinity towards the active site of the PD-L1 protein. In silico pharmacokinetic profiling of these screened compounds was done to test their biological activity. Next, experimental validation of the best four virtually screened hits was done in vitro for its hemocompatibility and cytotoxicity. Among these, Raltitrexed, Safinamide and Specs compound (AK-968/40642641) effectively increased the proliferation of immune cells and IFN-γ production. These compounds can act as potent PDL-1 inhibitors for adjuvant therapy against sepsis.

Abstract LB336: pH-sensitive polymeric micelles loading IL-12 profoundly inflame the tumor microenvironment to eradicate cold tumors

Abstract Background: Treatment of immunologically cold tumors is a major challenge. IL-12 can activates both innate and adaptive immunity to elicit strong antitumor immunity. Nevertheless, the poor pharmacokinetic and severe side effects of IL-12 limit the application. We have recently developed pH-sensitive polymeric micelles loading IL-12 (IL-12/m) that can selective activate in tumor tissues to invigorate immunotherapy and enhance efficacy. Herein, we elucidated the mechanisms behind the enhanced efficacy of IL-12/m and their ability to synergize with immune checkpoint inhibitors (ICIs). Methods: To determine the contribution of the immune cells on the efficacy on IL-12/m, mice bearing orthotopic 4T1 tumor models were treated with anti-CD4 (for depleting CD4+ cells), anti-CD8 (for depleting CD8+ cells), anti-asialo GM1 (for depleting NK cells). The tumor volume was tracked after treatment with IL-12/m. The immune status of the tumors was studied by histology, flow cytometry and RNA-seq after treatment with free IL-12, IL-12/m and their combination with ICIs. The efficacy of the treatments was assessed by following the tumor growth and mice survival. Results: IL-12 can promote the growth and function of T cells to initiate the enhanced anti-tumoral immune reactions. Flow cytometry analysis IL-12/m significantly enhanced tumor infiltration of CD8+ T cells compared to free IL-12. The immune cell depletion study revealed that the depletion of CD8+ cytotoxic T cells, CD4+ T cells and NK cells significantly lessen efficacy of IL-12/m, which is consistent with the biological function of IL-12. The depletion experiment suggests CD8+ cytotoxic T cells are the most important fraction for IL-12/m efficacy. Moreover, immunohistochemistry analysis of 4T1 revealed IL-12/m promoted high infiltration of CD8+ cells and the upregulation of Granzyme B in CD8 + Cells. IL-12/m also increased PD-L1 levels in tumors. The effects of IL-12/m on the TME was further investigated as a monotherapy and in combination with anti-PD-1 antibodies. The combination of IL-12/m with anti-PD-1antibodies presented high CD45+ cells in tumors. Lymphoid cells and T cells were upregulated by IL-12 and the anti-PD-1 antibody combination. The general CD4+ T cells also showed upregulation in the IL-12/m treated groups. While anti-PD-1 monotherapy did not show any suppression on Tregs in the tumor model, the combination of IL-12/m plus anti-PD-1 depleted Tregs. Moreover, IL-12/m increase the presence of Th cells. We then investigated the activation by transcriptome analysis. The heatmap of global gene expression indicated differentiated gene profiles. GOBP and KEGG analysis showed that IL-12/m upregulated the stimulation of proliferation, differentiation and activation of various effector immune cells, differentiation of T cells into Th cells, and T cell activation and T cell receptor signaling pathway. The enhanced the intratumoral immunity of IL-12/m plus ICIs allowed eradicating 4T1 tumors. Conclusions: Systemically injected IL-12/m ensured strong stimulation of antitumor immunity leading to high safety and strong therapeutic outcomes in cold tumor models. The enhanced immune response of IL-12 synergize with checkpoint blockade to eradicate cold tumors. The approach has potential for developing safer and more effective immunotherapies. Citation Format: Horacio Cabral, Pengwen Chen, Koji Nagaoka, Takuya Miyazaki, Kazunori Kataoka, Kazuhiro Kakimi. pH-sensitive polymeric micelles loading IL-12 profoundly inflame the tumor microenvironment to eradicate cold tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB336.

NLRP3 inhibitor dopamine receptors agonist as a potential therapeutic strategy for treatment of overactive immune responses in COVID-19

Patients infected with coronavirus disease 2019 (COVID-19) have high serum levels of proinflammatory cytokines. The “cytokine storm” has become one of the major causes of death for critically ill patients infected by COVID-19. Glucocorticoids, plasma from convalescent patients, blood purification, and tocilizumab are currently recommended for use when the body’s inflammatory response is overactivated. However, there are limitations in terms of medicinal effects, equipment reserves, and treatment expense. These challenges prompted us to assess classical agents with good safety and mature production technology. A recent study showed that nucleotide-binding oligomerization domain (NOD)-, leucine-rich repeat (LRR)-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes drive COVID-19 pathology. We speculate that suppression of NLRP3 inflammasome-derived cytokine production may be beneficial in COVID-19–infected patients. Dopamine receptors are present in almost all immune cells and can modulate their activation, proliferation, and cytokine production of immune cells. Previous studies have shown that dopamine receptor agonists can control systemic inflammation through inhibition of the NLRP3 inflammasome. This suggests that dopamine receptor agonists may be a new strategy for the treatment of overactive immune responses in COVID-19 patients. This is worthy of further investigation in clinical practice.

Phosphorylation of Tyrosine 841 Plays a Significant Role in JAK3 Activation.

Janus Kinase 3 (JAK3) plays a key role in the development, proliferation, and differentiation of various immune cells. It regulates gene expression by phosphorylation of Signal Transducers and Activators of Transcriptions (STATs) via the JAK/STAT pathway. Recently, we found a new JAK3 phosphorylation site, tyrosine 841 (Y841). The results showed that pY841 helps the kinase domain flip around the pseudo kinase domain, which may cause JAK3 conformational changes. It also reduces the size of the cleft between the N-lobe and the C-lobe of the JAK3 kinase domain. However, pY841 was found to enlarge the cleft when ATP/ADP was bound to the kinase. The increase in the cleft size suggested that pY841 enhanced the elasticity of the kinase domain. For unphosphorylated JAK3 (JAK3-Y841), the binding forces between the kinase domain and ATP or ADP were similar. After phosphorylation of Y841, JAK3-pY841 exhibited more salt bridges and hydrogen bonds between ATP and the kinase than between ADP and the kinase. Consequently, the electrostatic binding force between ATP and the kinase was higher than that between ADP and the kinase. The result was that compared to ADP, ATP was more attractive to JAK3 when Y841 was phosphorylated. Therefore, JAK3-pY841 tended to bind ATP rather than ADP. This work provides new insights into the role of phosphorylation in kinase activation and ATP hydrolysis and sheds light on the importance of understanding the molecular mechanisms that regulate the kinase function.

Abstract 1807: CB307: a novel targeted CD137 agonist for enhancement of immune cell responses to PSMA+ tumors

Abstract Introduction: CD137 (HGNC:TNFRSF9, 4-1BB) is a TNF receptor superfamily costimulatory receptor expressed by T cells and NK cells. Upon engagement, CD137 enhances immune cell survival, proliferation, cytokine production and memory formation. CD137 agonists in the clinic have shown tantalizing anti-tumor effects, however, hepatic toxicity has hampered clinical utility. To circumvent this limitation, we developed CB307, a half-life extended heavy-chain only Humabody® that selectively agonizes CD137 only in the presence of prostate specific membrane antigen (PSMA, HGNC:FOLH1). PSMA is highly upregulated on tumor cells in prostate cancer and tumor neovasculature in other solid tumors including lung (NSCLC), kidney (clear cell RCC), bladder and colorectal cancers. CB307 enhances immune cell activation in a PSMA-dependent manner. We present here preclinical mechanistic differentiators of CB307 with respect to non-targeted CD137 agonists. Methods: CD137 agonism, immune cell activation and cytokine secretion were evaluated in vitro using coculture assays in the presence/absence of PSMA-expressing tumor cells and in vivo with a transgenic mouse syngeneic tumor model. The ability of CB307 or non-targeted CD137 agonists to enhance tumor cell killing was additionally characterized in 3D spheroid cocultures. In vitro cocultures were used to determine the impact of PSMA expression level on CB307 activity. CB307 was evaluated as a single agent and in combination with PD-1 or PD-L1 inhibitors; tumoral checkpoint contribution was investigated using isogenic PD-L1 positive vs. PD-L1 CRISPR-deleted tumor cells. Results: CB307 or PD-1/PD-L1 inhibition alone induced IL-2 secretion in PBMC/PSMA-expressing tumor cell cocultures; this effect was synergistically enhanced when CB307 was combined with either PD-1 or PD-L1 inhibition. CB307 augmented tumor cell killing in 3D spheroids containing PSMA-expressing cells. Enhanced tumor cell killing was observed in spheroid cultures containing CB307 in combination with PD-1/PD-L1 inhibition. In in vivo pharmacology studies in a syngeneic mouse model, CB307 demonstrated statistically significant inhibition of tumor growth versus control without peripheral T cell activation - consistent with pharmacology restricted to the PSMA+ tumor microenvironment, whereas an anti-CD137 monoclonal antibody induced tumor growth inhibition accompanied by peripheral immune cell proliferation and cytokine production. Summary: CB307 is a novel CD137 agonist that selectively enhances immune cell activity only in the presence of PSMA-positive cells. Our data demonstrate PSMA-targeted CB307 immunological pharmacology both in vitro and in vivo which has supported the ongoing Phase 1 ‘POTENTIA’ clinical trial (NCT04839991). Citation Format: Andrew J. Pierce, Phillip M. Brailey, Sophie Archer, Clare Song, Anca-Giuilia Cionca, Joanne Fernando, Phillip D. Bartlett, Philip Bland-Ward. CB307: a novel targeted CD137 agonist for enhancement of immune cell responses to PSMA+ tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1807.

Abstract 971: Thymidine Kinase activity - A liquid biomarker reflecting proliferation rate and treatment efficacy in solid tumors provides unique guidance in oncology trials and drug development

Abstract Thymidine Kinase (TK) plays a fundamental role in DNA synthesis and cell proliferation. Expression of the enzyme is highly cell cycle dependent and correlates with cell growth state. TK activity (TKa) is present in all proliferating cells but release into blood circulation is low or absent from quiescent cells. Numerous studies have shown TKa to be elevated in solid tumors as well as in the blood of cancer patients with active disease. DiviTum® TKa, a clinically and analytically validated FDA cleared assay, can measure and quantify TKa in serum or plasma isolated from human and animal blood samples. TKa as a biomarker offers valuable information at all stages of drug development; from drug efficacy and dose response studies in cell culture and tumor xenograft models, to patient selection and treatment monitoring in clinical trials. Using TKa to measure the impact on cell proliferation as a drug evaluation endpoint can confirm mechanism of action efficacy and the minimally effective dose of a new compound rather than a maximally tolerated dose. In both cultured cells and mouse xenografts, a complete shutdown of tumor cell proliferation (assessed by TKa) can be observed well before and at much lower doses than when cell death or toxicity is used as the dose determination endpoint. The TKa-biomarker can also be used as a pharmacodynamic tool in clinical trials to improve study endpoints. Patient baseline levels (BL) of TKa are predictive/prognostic and can be used to identify which patients will respond to the therapy being tested vs those who will not. In metastatic breast cancer, a BL TKa value of <250 DuA is significantly correlated with longer PFS (HR=1.76, p<0.0001) and OS (HR=2.38, p<0.0001) independent of patient and disease characteristics. On-therapy TKa levels and dynamics can be used in clinical trials to evaluate response to a drug as early as two weeks into the first cycle. In patients receiving CDK4/6 inhibitors, TKa levels >140 DuA at cycle 1 day 15 predicts for lack of benefit to the drug (HR=5.65, p<0.0001). TKa can also be used as a predictive biomarker for drugs that cause an immune reaction and increase in cell proliferation such as immune checkpoint inhibitors (ICIs). When effective, ICIs cause an increase in immune cell proliferation which can be detected by elevated levels of TKa. Patients who do not achieve >1.5 fold change in TKa levels as compared to BL, have a significantly shorter duration of response to an ICI (HR=3.70, p=0.002) and patients with high TKa during ICI treatment have significantly longer OS (HR=3.49, p<0.05). In summary, TKa is a translational liquid biomarker that can bridge between preclinical and clinical studies, providing fundamental information for decision making during early and late stage drug development. Data and examples of the utility of TKa as a companion diagnostic for drug development will be presented. Citation Format: Mattias K. Bergqvist, Hanna Ritzén, Amy Williams. Thymidine Kinase activity - A liquid biomarker reflecting proliferation rate and treatment efficacy in solid tumors provides unique guidance in oncology trials and drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 971.

Abstract 4113: Classification of patient-specific sensitivity to immunotherapies by ex vivo tumor testing

Abstract Background: Immunotherapy has brought great progress with durable responses for cancers that were difficult to treat. However, it remains challenging to select sensitive patients upfront. PD-L1 expression, TMB and MSI/MSS status, do not optimally differentiate between the potential sensitivity to the individual immunotherapies. Purpose: We aim to improve stratification of patients for immunotherapy through classification of patient-specific tumor sensitivity based on 3D ex vivo drug measurement. Material: Our study includes 108 Patients with predominantly ovarian carcinoma (81/108). Tumor tissue was collected in ongoing clinical trials from biopsies, ascites or pleural fluid. Five immune checkpoint inhibitors and a STING pathway activator were tested. Method: Fresh tumor clusters were seeded into 384 well plates and exposed to different immunotherapies, while preserving the TME. Tumor killing and immune cell proliferation were measured using 3D image analysis. Ex vivo tumor sensitivity was classified as no response (<10%), weak (10-20%), strong (20-50%), and very strong (>50%), based on percentage tumor killing and statistical significance (p-value: * < 0.05, ** < 3.33e-4). Results: Differential patient response profiles were observed (Table 1). For the current cohort, we measured a highly significant immunotherapy response for ~30% of the samples. The assay has a technical success rate of 89%. Conclusion: This study reports the development of a robust ex vivo tumor testing platform that classified patient-specific sensitivity to 6 immunotherapies for over 100 patients, demonstrating the potential of ex vivo tumor testing to optimize patient stratification for immunotherapy. Discussion: The platform enables improved efficiency in clinical development of novel treatments and supports treatment decisions in the clinic. Clinical trials are ongoing to establish the correlation of our testing with patients' response to immunotherapy in lung cancer. Overview of ex vivo immunotherapy response per category 1Therapy # Samples tested % Response No response Weak Strong Very strong SEA (control) 108 43% (30%**) 62 5 19 22 CD3/CD28 Dynabeads (control) 18 33% (17%**) 12 1 5 0 Pembrolizumab 89 16% (8%**) 75 6 5 3 Nivolumab 72 10% (4%**) 65 1 5 1 Ipilimumab 97 20% (11%**) 78 4 10 5 Atezolizumab 62 15% (11%**) 53 1 4 4 Durvalumab 52 2% (0%**) 51 1 0 0 ADU-S100 60 37% (27%**) 38 5 9 8 Citation Format: Lieke Johanna Ceton, Marta Garcia Montero, Fanny Grillet, Dieudonné J. van der Meer, Willemijn Vader, Anne van Altena, Cor D. de Kroon, Nelleke Ottevanger, Judith Kroep. Classification of patient-specific sensitivity to immunotherapies by ex vivo tumor testing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4113.

Abstract 4570: Expanding the 3D ex vivo patient tissue platform toolbox for (immuno)-oncology drug testing

Abstract Introduction: The fast-paced (immuno)-oncology drug development pipeline requires sophisticated and reliable in vitro models to accurately represent all players of complex human tumor microenvironment (TME). Our 3D Ex Vivo Patient Tissue (EVPT) platform is a short-term 3D ex vivo culture system utilizing ultra-fresh human tumor material processed to preserve the rich TME with high content image (HCI) analysis for higher throughput screening. The EVPT platform has previously been validated for standard of care (SoC) and immunological drug testing, including immune checkpoint inhibitors (ICI), in ovarian and non-small cell lung cancer (NSCLC) samples. Here, we present the expansion of the EVPT platform to other cancer indications, including resections from bladder, prostate, breast cancer, melanoma, glioblastoma (GBM) and hepatocellular carcinoma (HCC). Methods: Tumor tissues from NSCLC, ovarian, bladder, prostate, breast cancer, melanoma, GBM and HCC patients were processed within 24 hours. Freshly isolated tumor clusters were embedded in a protein-rich hydrogel and exposed to panels of (immuno)-oncology treatments at various doses in a 384-well format for 5-7 days. Phenotypic effects of treatment on morphological features, such as tumor cell killing, growth arrest, and immune cell proliferation, were measured using our proprietary automated HCI platform. Whole blood samples were also collected and PBMCs were isolated for autologous co-culture assays with selected cancer samples. Additionally, biomarkers analysis, including IHC, FACS and cytokine measurements, was performed. Results: At least two patient samples per new indication were processed. The success rate of the assay (30-40%) was primarily dependent on the sample quality, and the absence of necrotic, healthy, or fibrotic tissue within the sample. Panels of SoC drugs, targeted therapies, immunotherapies, and control compounds established for each indication were profiled per patient sample. For example, prostate cancer samples responded to Paclitaxel and Crizotinib, and one of GBM samples showed sensitivity to Regorafenib and Bevacizumab. Overall, similarly to clinical findings, about 20% of NSCLC and bladder cancer samples responded to ICI treatment, while 60-70% of the samples have responded to immunostimulatory controls (SEA and CD3/CD28).Although TME composition varied between NSCLC samples, IHC and FACs analysis showed a rich representation of immune compartment - T Cells (CD3+CD4+, CD3+CD8+), NK cells (CD16+), myeloid cells (CD14+, CD68+) alongside with EpCAM+ tumor cells. Conclusion: Despite tissue quality remaining a challenge for many primary ex vivo assays, the gentle processing and short-term 3D culturing in our EVPT platform preserves the rich TME in a wide range of tumors. This makes our EVPT platform an innovative and translational tool for complex (immuno)-oncology drug testing in multiple cancer indications. Citation Format: Nataliia Beztsinna, Sander Basten, Ezgi Kaya Aksoy, Niels Meesters, Stefanos Timiliotis, Donny van der Meer, Kuan Yan, Emma Spanjaard, Willemijn Vader, Leo S. Price. Expanding the 3D ex vivo patient tissue platform toolbox for (immuno)-oncology drug testing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4570.

Abstract 2109: Analytical characterization of a multiplex panel measuring 45 cytokines by proximity extension assay in plasma from subjects with NSCLC

Abstract Introduction Cytokines are fundamental regulators of immune system processes and are responsible for the differentiation, proliferation, and function of immune cells. With many anti-cancer therapeutic strategies focused on targeting or harnessing the immune system, or components thereof, quantification of circulating cytokines has become important to describe the pharmacodynamic and mechanistic effects of these investigational therapies. To support these efforts, new multiplexed immunoassay panels have been developed to expand the available options for broad spectrum characterization of cytokine profiles from biospecimens. Here we characterize the analytical performance of one such panel based on proximity extension assay technology to measure 45 cytokines in plasma. Experimental Procedures Plasma samples (n = 27) from patients with non-small cell lung cancer (NSCLC), along with age-matched plasma samples (n = 6) from healthy normal subjects were analyzed using the Olink Cytokine 48 kit. Samples were thawed and processed according to manufacturer instructions for the incubation, extension, and detection on the Olink Signature Q100 instrument. A subset of the screened samples was run in quadruplicate across multiple runs and analyzed on two separate instruments. Results Of the 45 cytokines assayed, 41 had values above the assay limit of detection in ≥75% of samples analyzed. When compared to the normal human plasma, 26 cytokines were significantly elevated (p-value ≤ 0.05) in plasma from NSCLC subjects, including those known to be associated both with poor (e.g., IL-1β, IL-6) and good prognosis (e.g., IFN-γ). Furthermore, many of the cytokines with significant differences were of low abundance; 13 of the 26 cytokines had mean values ≤ 20.0 pg/mL. For two of the incurred samples which were run across multiple replicates and runs, with mid- to high- levels of cytokines, the median intra-assay CV across all assays was 6.2% with 90% of the assays demonstrating CVs of less than 12%. This only increased to a 7.7% median CV in inter-run comparison with 90% of the assays remaining below 12%. Additional results from the incurred sample analysis will be presented. Conclusions These results demonstrate that multiplex cytokine analysis, based on proximity extension assays, can be effectively deployed to screen a large number of targets and can uncover clinically relevant changes in low abundant cytokines. Citation Format: Anne Jang, Daniel Feingold, Mark H. Watson, Gwenaël Pottiez, Rudolf Guilbaud, Nicholas Francois Dupuis. Analytical characterization of a multiplex panel measuring 45 cytokines by proximity extension assay in plasma from subjects with NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2109.

Abstract 4234: Effects of age-diet interactions on colorectal cancer (CRC) progression in mice

Abstract Background. CRC is the third leading cause of cancer related deaths worldwide with age and diet are among the strongest risk factors. CRC characteristics appear to be distinct between younger and older patients. We evaluated the effects of age on CRC tumor biology using syngeneic mice models with a focus on a) specific differences in tumor growth patterns and the role of the tumor microenvironment (TME) in young versus old mice harboring CRC allografts; b) differential dietary effects in young versus old hosts. Methods. 1 × 106 MC38 cells were subcutaneously implanted into the right flank of young (6 weeks) or old (24 months) C57BL/6 male mice. Mice were randomized into 3 diet groups: A. Standard Chow (SC); B. Calorie Restriction (CR: 30% reduction in total calories); C. High Fat (HF). Parameters studied were daily food intake, body weight, tumor growth and survival. Once endpoint reached, tumors were isolated and processed for RNA sequencing. Cytokine analysis was done on plasma using multiplex immunoassays on the Meso Scale Discovery platform. Results. Both old and young mice showed significant reduction in tumor growth while on the CR protocol when compared to other dietary groups. Young mice fed with HF diet had increased tumor growth as compared to old. Cytokine analysis showed that the pro tumorigenic IFNγ, TNF α, IL10, IL-12p70, and 1L6 were higher in old mice relative to young. Caloric restriction irrespective of age led to significant reductions in IFNγ, TNFα, and 1L6 compared to other diets. Irrespective of diet, IL10, known for its immunosuppressive role in CRC, was significantly lower in young versus old mice. Proinflammatory IL-5 was significantly higher in young fed a HF diet compared to the other groups. RNA sequencing of the allograft tumors at the end of the study revealed that differentially expressed genes (DEGs) between young and old mice tumors were dramatically influenced by diet. The most affected gene expression pathways were antigen processing and presentation, MHC-II complex assembly and T cell activation. Diet affected the tumor expression of key oncogenes associated with immune escape, cell proliferation, invasion, metastasis, chemotaxis, ECM remodeling and chemosensitivity which were upregulated in old mice and HF diet. Conclusion. Based on transcriptomic profiles and comprehensive cytokine analysis, our data shows that age-diet interactions significantly influence the host TME leading to differential gene expression in the tumor. Our findings will advance current understanding of the mechanisms by which aging and diet impact CRC onset, progression, metastasis, and therapeutic response but warrants further investigation. Citation Format: Jae Ho Lo, Shivani Soni, Yan Yang, Goar Smbatyan, Junxiang Wan, Joshua Millstein, Kelvin Yen, Hemal H. Mehta, Brendan Miller, Francesca Battaglin, Pooja Mittal, Lesly Torres Gonzalez, Wu Zhang, Pinchas Cohen, Heinz Josef Lenz. Effects of age-diet interactions on colorectal cancer (CRC) progression in mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4234.

Abstract 1809: ACT-1002-4391 - A novel potent antagonist of the prostaglandin E2 receptors EP2 and EP4 with excellent duality

Abstract Prostaglandin E2 (PGE2) binds to four G-protein-coupled EP receptors, of which both EP2 and EP4 are abundantly expressed by tumor-infiltrating immune cells including DCs, NK cells, Tregs, cytotoxic T cells and TAMs. Signaling via EP2 and EP4 is immunosuppressive, preventing the mounting of effective anti-tumor immune responses leading to tumor immune escape and tumor progression. Currently, several EP4 selective antagonists and an EP2/4 dual antagonist are being developed for anti-cancer immunotherapy. Using a combination of EP2 and EP4 selective antagonists in different tumor mouse models, we could demonstrate that dual EP2/EP4 blockade is more effectively activating anti-tumor immunity compared to targeting either receptor alone. The concurrent inhibition of EP2 and EP4 resulted in tumor regression and induction of immunological memory. Together with published data showing similar findings, our results suggest that simultaneous targeting of EP2 and EP4 could be a promising new therapeutic approach to overcome cancer immune evasion. Idorsia Pharmaceuticals has identified ACT-1002-4391 as a novel, potent antagonist of EP2 and EP4 receptors with an excellent duality profile. In vitro, ACT-1002-4391 is capable of modulating immune cell proliferation and/or function and is superior to EP2 or EP4 selective inhibition in that respect. In vivo, ACT-1002-4391 displays strong and dose-dependent single agent anti-tumor efficacy in a syngeneic mouse tumor model. Based on the excellent selectivity and duality profile, ACT-1002-4391 will be advanced into clinical development by Idorsia Pharmaceuticals. Citation Format: Sébastien Jeay, Stefan Diethelm, Agnes Knopf, Marie Goxe, Marie Daugan, Julie Erupathil, Julien Pothier, Davide Pozzi, Thierry Sifferlen, Lorenza Wyder, Isabelle Lyothier, Olivier Corminboeuf, Francois Lehembre, Imke Renz, Dominique Meyer. ACT-1002-4391 - A novel potent antagonist of the prostaglandin E2 receptors EP2 and EP4 with excellent duality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1809.

Abstract 1781: G9a/GLP inhibition during ex vivo lymphocyte expansion increases in vivo cytotoxicity of engineered T cells against hepatocellular carcinoma

Abstract Adoptive cell transfer (ACT) has been highly efficient in targeting certain refractory cancers and remains potentially effective for other cancers as new targets of cancer-immune interactions are revealed. ACT involves isolating immunocompetent cells from cancer patients, expanding them ex vivo, and infusing them back into the patient. Cells used for ACT are often effector cells, typically T cells, isolated from the patient’s peripheral blood and then engineered to target cancer cells by incorporating a T cell receptor (TCR) or chimeric antigen receptor (CAR), with additional modifications to improve immune cell proliferation and persistence. While cell therapies for various lymphomas have led to dramatic tumor regressions, response in solid tumors remains varied. Solid tumors present unique challenges, such as tumor heterogeneity and the lack of tumor-specific targets, but also an immunosuppressive tumor microenvironment (TME) characterized by poor T cell infiltration and terminal T cell differentiation and exhaustion at the tumor. To overcome the hostile TME, lymphodepleting regimens combined with an infusion of large numbers of T cells is often used. However, introducing large numbers of T cells significantly increases the risk of on-target/off-target toxicity, neurotoxicity, and cytokine release syndrome. The use of engineered T cells that transiently express TCRs or CARs by mRNA gene transfer, reduces these risks. Additionally, they avoid the use of viral vectors, hence there is no risk of insertional mutagenesis and can be manufactured more easily at low cost and on a larger scale. Such transient-engineered T cells have shown significant antitumor activity in phase I clinical trials and in preclinical animal models. However, multiple infusions are often required, increasing patient discomfort and treatment cost. To mitigate this, we sought to improve the antitumor activity of transient engineered T cells by screening a panel of small molecules targeting epigenetic regulators for their effect on T cell cytotoxicity. Using a model for engineered T cells targeting hepatocellular carcinoma, we found that short-term inhibition of G9a/GLP increased T cell antitumor activity in in vitro models and an orthotopic mouse model. G9a/GLP inhibition increased granzyme expression without terminal T cell differentiation or exhaustion and resulted in specific changes in the expression of genes and proteins involved in pro-inflammatory pathways, T cell activation, and cytotoxicity. Citation Format: Maxine S. Lam, Jose A. Reales-Calderon, Jin Rong Ow, Joey Aw, Damien Tan, Ragavi Vijayakumar, Erica Ceccarello, Tommaso Tabaglio, Yan Ting Lim, Wang Loo Chien, Fritz Lai, Anthony Tan Tanoto, Qingfeng Chen, Radoslaw M. Sobota, Giulia Adriani, Antonio Bertoletti, Ernesto Guccione, Andrea Pavesi. G9a/GLP inhibition during ex vivo lymphocyte expansion increases in vivo cytotoxicity of engineered T cells against hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1781.

Ubiquitin-Specific Protease 43 Impacts Pancreatic Ductal Adenocarcinoma Prognosis by Altering Its Proliferation and Infiltration of Surrounding Immune Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer, and the therapy options for PDAC remain restricted. The distinctive tumor immunological microenvironment (TIME) of PDAC, comprising a high number of stromal cells and a limited infiltration of cytotoxic T lymphocytes (CTLs), rendered immunotherapy ineffective. The protein level of ubiquitin-specific protease 43 (USP43) was a prognostic predictor in numerous cancers; however, its function in PDAC is limited. This article focuses on the influence of USP43 expression on PDAC prognosis and TIME alteration. Based on TCGA database and tissue microarray staining, the expression of USP43 in PDAC was evaluated. The association between USP43 and prognosis was then investigated using tissue samples and online databases. In PDAC tumor tissues, the correlation between USP43 expression and clinicopathological characteristics, immune cell infiltration, and prognosis was investigated. The expression of USP43 in PDAC cell lines was evaluated using quantitative polymerase chain reaction. Using a cell counting kit-8 (CCK-8) and a cell colony formation test, the viability of the cells was determined. On the basis of online databases and tissue samples, the link between USP43 and immune cell infiltration around PDAC was also examined. For statistical analyses, the software GraphPad, R, and SPSS 26.0 were utilized. The expression of USP43 was considerably higher in PDAC compared to normal pancreatic tissue in both the TCGA database and the tissue microarrays of PDAC patients (P < 0.001). High USP43 expression was associated with poor overall survival in both the TCGA database and the tissue microarray of PDAC patients (P = 0.046 and 0.021, respectively). USP43 overexpression promoted PANC-1 cell proliferation (P = 0.0018), but USP43 knockdown decreased PANC02 cell proliferation (P < 0.001). According to the TCGA database, USP43 is associated with T cell activation and inhibits CD8+ T cell activation in PDAC, as proven by a study of cell lines. Moreover, in both TCGA and PDAC cell lines, USP43 expression was negatively linked with the chemokine signaling pathway. Overexpression of USP43 is a potential prognostic indicator for PDAC patients. USP43 is a potential biomarker associated with T cell activation, suppression of CD8+ T cell enrichment, and the cytokine signal pathway. Future multicenter studies are needed to confirm our findings and their potential application in the treatment of PDAC patients.

MicroRNAs in inflammasomopathies

microRNAs (miRNAs) are small non-coding RNA sequences that negatively regulate the expression of protein-encoding genes at the post-transcriptional level. They play a role in the regulation of inflammatory responses by controlling the proliferation and activation of immune cells and their expression is disrupted in several immune-mediated inflammatory disorders. Among these, autoinflammatory diseases (AID) are a group of rare hereditary disorders caused by abnormal activation of the innate immune system and characterized by recurrent fevers. Major groups of AID are inflammasomopathies, which are associated with hereditary defects in the activation of inflammasomes, cytosolic multiprotein signaling complexes regulating IL-1 family cytokine maturation and pyroptosis. The study of the role of miRNAs in AID is only recently emerging and remains scarce in inflammasomopathies. In this review, we describe the AID and inflammasomopathies, and the current knowledge on the role of miRNAs in disease processes.

Identification of the key miRNA-mRNA regulatory network in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most common type of lung cancer, has a high incidence rate and is a serious threat to human health. However, the pathogenesis of LUAD is still unclear. Further research on the pathogenesis of LUAD may provide targets for the early diagnosis and treatment of LUAD. First, a transcriptome analysis was conducted to sequence the messenger RNA (mRNA) and micro RNA (miRNA) of the LUAD and adjacent control tissues. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were then performed for the functional annotation. Next, a differential miRNA-differential mRNA regulatory network was then constructed, and the function of the mRNAs in the network was analyzed and the key regulatory molecules (the hub molecules) were identified. Cytohubba was then used to analyze the top 20 hub molecules in the total miRNA-mRNA network, and the miRNAs regulating the top 20 hub genes (of which 2 were upregulated and 18 were downregulated). Finally, the key molecules were identified. By analyzing the function of the mRNA molecules in the regulatory network, we found that the immune response was inhibited, as were the movement and adhesion of immune-related cells; however, cell tumorigenesis, body death, and tumor cell proliferation were activated. The functions of the 20 hub molecules were mainly related to cytotoxicity, cell exosmosis, and cell adhesion mediated by immune cells. Further, we found that miR-5698, miR224-5p, and miR4709-3P regulate multiple key genes (e.g., PECAM1, CX3CR1, KLRD1, and CXCL12), and may be the key miRNAs regulating LUAD. Immune response, cell tumorigenesis, and tumor cell proliferation play central roles in the overall regulatory network. miR-5698, miR224-5p, and miR4709-3p may be important biomarkers for the occurrence and development of LUAD and have great potential in the prognosis of LUAD patients and the development of new therapeutic targets.

How Staphylococcus aureus and Pseudomonas aeruginosa Hijack the Host Immune Response in the Context of Cystic Fibrosis.

Cystic fibrosis (CF) is a serious genetic disease that leads to premature death, mainly due to impaired lung function. CF lungs are characterized by ongoing inflammation, impaired immune response, and chronic bacterial colonization. Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) are the two most predominant bacterial agents of these chronic infections. Both can colonize the lungs for years by developing host adaptation strategies. In this review, we examined the mechanisms by which SA and PA adapt to the host immune response. They are able to bypass the physical integrity of airway epithelia, evade recognition, and then modulate host immune cell proliferation. They also modulate the immune response by regulating cytokine production and by counteracting the activity of neutrophils and other immune cells. Inhibition of the immune response benefits not only the species that implements them but also other species present, and we therefore discuss how these mechanisms can promote the establishment of coinfections in CF lungs.

Nicotine Reduces Reactive Oxygen Species and Enhances Cell Proliferation via the α4 Nicotinic Acetylcholine Receptor Subunit in Human Induced Pluripotent Stem Cells.

The effects of smoking on fetal development and stem cell differentiation are not fully understood. Although nicotinic acetylcholine receptors (nAChRs) are expressed in many organs of the human body, their significance in human induced pluripotent stem cells (hiPSCs) remains unclear. After expression levels of nAChR subunits in hiPSCs were determined, the effects of the nAChR agonist, nicotine, on undifferentiated hiPSCs were evaluated using a Clariom S Array. We also determined the effect of nicotine alone and with a nAChR subunit antagonist on hiPSCs. nAChR α4, α7, and β4 subunits were strongly expressed in hiPSCs. cDNA microarray, gene ontology, and enrichment analyses showed that exposing hiPSCs to nicotine altered expression of genes associated with immune responses, neurological system, carcinogenesis, cell differentiation, and cell proliferation. Particularly affected was metallothionein, which acts to decrease reactive oxygen species (ROS). The nicotine-induced reduction of ROS in hiPSCs was canceled by an α4 subunit or nonselective nAChR antagonist. HiPSC proliferation was increased by nicotine, and this effect, too, was canceled by an α4 antagonist. In conclusion, nicotine reduces ROS and enhances cell proliferation through the α4 nAChR subunit in hiPSCs. These findings provide new insight into the significance of nAChRs on human stem cells and fertilized human ova.