Immune Cell Proliferation Research Articles

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.

Network Pharmacology and Molecular Docking Analysis of the Mechanisms of Combined Radix Bupleuri (Chai-Hu) and Radix Paeoniae Alba (Bai-Shao) Treatment in the Prevention and Treatment of Alzheimer’s Disease

Abstract Objective: Radix Bupleuri and Radix Paeoniae Alba are referred to as Chai-Hu and Bai-Shao, respectively, in Chinese. We used molecular docking and network pharmacological approaches to explore the active components of this Chinese herb combination which is commonly used for treating Alzheimer’s disease (AD), the Chai-Hu and Bai-Shao pair (CBP). Materials and Methods: The active compounds and action targets of Chai-Hu and Bai-Shao were identified in the Traditional Chinese Medicine Systems Pharmacology database, and the Therapeutic Target Database, DrugBank, GenCards, DisGeNET, and Online Mendelian Inheritance in Man databases were used to identify therapeutic targets in patients with AD. To identify a common target of CBP in the treatment of AD, we used the Metascape tool to perform Gene Ontology function enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. In addition, CBP-AD protein–protein interaction (PPI) and herb-component-target networks were created using Cytoscape 3.8.2. Molecular docking was verified using AutoDock tools. Results We identified 20 active compounds of CBP, 202 active targets, 1,323 disease targets, and 117 active compound disease intersection targets; the PPI network showed that the top eight key targets were AKT1, interleukin (IL) 6, JUN, MAPK1, tumor necrosis factor, TP53, vascular endothelial growth factor, and epidermal growth factor. These act mainly on the fluid shear stress, atherosclerosis, IL-17, hypoxia-inducible factor-1, and nonalcoholic fatty liver disease pathways, among others, which control immune inflammation, cell proliferation, and apoptosis. Based on molecular docking results, the active components of CBP bind well to its key targets. Conclusion Our results indicated that CBP was effective for treating AD by reducing the expression of inflammatory factors, promoting cell proliferation and differentiation, and preventing nerve cell death and anti-neuronal cell apoptosis.

Role of gut microbiota in infectious and inflammatory diseases

Thousands of microorganisms compose the human gut microbiota, fighting pathogens in infectious diseases and inhibiting or inducing inflammation in different immunological contexts. The gut microbiome is a dynamic and complex ecosystem that helps in the proliferation, growth, and differentiation of epithelial and immune cells to maintain intestinal homeostasis. Disorders that cause alteration of this microbiota lead to an imbalance in the host's immune regulation. Growing evidence supports that the gut microbial community is associated with the development and progression of different infectious and inflammatory diseases. Therefore, understanding the interaction between intestinal microbiota and the modulation of the host's immune system is fundamental to understanding the mechanisms involved in different pathologies, as well as for the search of new treatments. Here we review the main gut bacteria capable of impacting the immune response in different pathologies and we discuss the mechanisms by which this interaction between the immune system and the microbiota can alter disease outcomes.

Oxidative Stress and Epigenetics: miRNA Involvement in Rare Autoimmune Diseases.

Autoimmune diseases (ADs) such as Sjögren's syndrome, Kawasaki disease, and systemic sclerosis are characterized by chronic inflammation, oxidative stress, and autoantibodies, which cause joint tissue damage, vascular injury, fibrosis, and debilitation. Epigenetics participate in immune cell proliferation and differentiation, which regulates the development and function of the immune system, and ultimately interacts with other tissues. Indeed, overlapping of certain clinical features between ADs indicate that numerous immunologic-related mechanisms may directly participate in the onset and progression of these diseases. Despite the increasing number of studies that have attempted to elucidate the relationship between miRNAs and oxidative stress, autoimmune disorders and oxidative stress, and inflammation and miRNAs, an overall picture of the complex regulation of these three actors in the pathogenesis of ADs has yet to be formed. This review aims to shed light from a critical perspective on the key AD-related mechanisms by explaining the intricate regulatory ROS/miRNA/inflammation axis and the phenotypic features of these rare autoimmune diseases. The inflamma-miRs miR-155 and miR-146, and the redox-sensitive miR miR-223 have relevant roles in the inflammatory response and antioxidant system regulation of these diseases. ADs are characterized by clinical heterogeneity, which impedes early diagnosis and effective personalized treatment. Redox-sensitive miRNAs and inflamma-miRs can help improve personalized medicine in these complex and heterogeneous diseases.

T-Cell Repertoire Analysis in the Conjunctiva of Murine Dry Eye Model.

Dry eye is closely related to the activation and proliferation of immune cells, especially T cells. However, the determination of the preferential T-cell clonotypes is technically challenging. This study aimed to investigate the characterization of T-cell receptor (TCR) repertoire in the conjunctiva during dry eye. A desiccating stress animal model was established using C57/BL6 mice (8-10 weeks, female). After 7 days of stress stimulation, the slit-lamp image and Oregon-green-dextran staining were used to evaluate the ocular surface injury. Periodic acid-Schiff staining was used to measure the number of goblet cells. Flow cytometry was used to detect the activation and proliferation of T cells in the conjunctiva and cervical lymph nodes. Next-generation sequencing was used to detect the αβ TCR repertoire of the conjunctiva. The αβ TCR diversity increased significantly in the dry eye group, including the higher CDR3 amino acid length, marked gene usage on TCR V and J gene segments, extensive V(D)J recombination, and distinct CDR3 aa motifs. More important, several T-cell clonotypes were uniquely identified in dry eye. Furthermore, these perturbed rearrangements were reversed after glucocorticoid administration. A comprehensive analysis of the αβ TCR repertoire in the conjunctiva of the dry eye mouse model was performed. Data in this study contributed significantly to the research on dry eye pathogenesis by demonstrating the TCR gene distribution and disease-specific TCR signatures. This study further provided some potential predictive T-cell biomarkers for future studies.

Abstract P2-20-06: Anti-B7-H3 Antibody (T-1A5) Blocks Immunomodulatory Function of B7-H3 and Enhances NK and T Cell–Mediated Cytotoxicity against Breast Cancer Cells

Abstract One of the main factors contributing to breast cancer (BC) initiation and metastasis is immune suppression by tumor cells. Immune checkpoint blockade has recently been shown to overcome tumor-induced immune suppression, but a significant proportion of patients do not respond, implying that more effective cancer immunotherapies are required. B7-H3 belongs to B7 family of immune checkpoint proteins that is overexpressed in various human malignancies. Here, we hypothesize that blocking B7-H3 using monoclonal antibodies (mAbs) enhances immune cell proliferation and function. To test our hypothesis, B7-H3 expression was determined using RNA-seq data from primary and metastatic breast tumors, and adjacent normal tissues, from the TCGA and METABRIC databases. To assess B7-H3 protein expression in BC, we performed immunohistochemistry (IHC) on frozen primary tumor tissues (n=50) and adjacent normal tissues (n=23) from TNBC patients. In addition, to investigate the immunomodulatory effect of B7-H3 in BC, we performed IHC for T-cell markers in subsets of patient tissues with high and low levels of B7-H3 expression. Next, we assessed B7-H3 expression in over 13 BC cell lines, including TNBC and ER+, PR+, and HER2+ cell lines, as well as TNBC PDX-derived cells. Moreover, to determine the effect of B7-H3 knockdown on NK- and T-cell activity, we co-cultured control and B7H3–KD BC cells in the presence and absence of NK and T cells and measured the induction of apoptosis in BC cells through IncuCyte live-cell imaging system. The effect of a novel B7H3-blocking mAb (clone T-1A5, isotype IgG1) on NK-cell and T-cell-mediated cytotoxicity in BC cell lines was examined using live-cell imaging. In the TCGA and METABRIC databases, B7-H3 was found to be significantly overexpressed (P&amp;lt; 0.0001) in tumor tissues than in adjacent normal tissues of BC patients. A survival analysis by the log-rank test indicated that patients with B7-H3high tumors had significantly lower progression-free (P=0.01) and relapse-free (P=0.0026) survival than patients with B7-H3low tumors. Moreover, B7-H3 is significantly upregulated in all the BC subtypes including basal, luminal A, luminal B and Her2-enriched with highest expression in basal type BC. Relative mRNA quantification and flow cytometry analysis demonstrated strong B7-H3 expression in most of the BC cell lines including TNBC and ER+, PR+, and HER2+ cell lines, as well as TNBC PDX-derived cells. Furthermore, IHC analysis revealed that compared to the matched normal tissue, B7-H3 expression was substantially higher in tumor tissue (N=16, P&amp;lt; 0.001). Also, patients with high B7-H3 expression had significantly lower numbers of CD3+, CD4+, and CD8+ T-cell, compared to patients with low B7-H3 expression (P&amp;lt; 0.001), indicating immunosuppressive role of B7-H3. Furthermore, NK cell and T cell mediated killing was significantly higher in B7H3-KD BC cell lines compared to control cells. We observed a significant increase in the killing of BC cells by NK cells and T cells in the presence of anti-B7H3 mAb T-1A5 in a concentration dependent manner (P &amp;lt; 0.001), suggesting that anti-B7H3 antibodies suppress the immunomodulatory function of B7-H3 and enhance NK and T cell–mediated ADCC in BC. To determine the antibody-dependent cell-mediated cytotoxicity, we developed a human-mouse chimera of T-1A5 (chT-1A5) and tested in combination with NK cells. Interestingly, we found a concentration and time dependent increase in ADCC in BC cells in the presence of chT-1A5 antibody. Our data suggests that B7-H3 is overexpressed in primary BC and inhibits immune-cell infiltration. Moreover, blocking the immunomodulatory functions of B7-H3 using anti-B7H3 antibody T-1A5 enhances NK and T cell–mediated killing of BC cells. Citation Format: Vivek Anand, Anudishi Tyagi, Venkata Lokesh Battula. Anti-B7-H3 Antibody (T-1A5) Blocks Immunomodulatory Function of B7-H3 and Enhances NK and T Cell–Mediated Cytotoxicity against Breast Cancer Cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-06.

The Effects of Diisononyl Phthalate Exposure on Colorectal and Leukemia Cancer Cell

Phthalates (PAEs), also known as phthalates, are collectively known as phthalimide esters and are mainly used as plasticizers (plasticizers), added to plastics to enhance elasticity, transparency, durability and longevity. Diisononyl phthalate (DiNP), a type of PAEs, is commonly used in a large variety of plastic items, and is listed as a substance "known to the State of California to cause cancer" under Proposition 65 legislation. PVC plastics have become ubiquitous in our modern society. DiNP leaches from PVC-based consumables, eventually leading to deposition. DiNP leached from consumables can eventually be deposited in certain tissues through inadvertent application. Studies on cancer cells leukemia cancer cells and colorectal cancer cells have demonstrated health risks in human populations exposed to DiNP, including endocrine disorders, reproductive damage, and tumorigenesis. In this research, we studied the effect DiNP causes on the leukemia cancer cell and colorectal cancer cell. The results show that DiNP has a proliferative effect on immune cells and an inhibitory effect on colorectal cancer cells. We do not know what specific pathway or mechanism DiNP could activate; However, it is highly likely that DiNP causes damage because it can trigger the proliferation of immune cells, or lesions, by modulating some of the organelles or receptors in immune cells. This effect is beneficial in non-immune cancers, but harmful in cancers that’s immune cell related, in this case, leukemia.

Tumor Radiosensitization by Gene Electrotransfer-Mediated Double Targeting of Tumor Vasculature.

Targeting the tumor vasculature through specific endothelial cell markers involved in different signaling pathways represents a promising tool for tumor radiosensitization. Two prominent targets are endoglin (CD105), a transforming growth factor β co-receptor, and the melanoma cell adhesion molecule (CD1046), present also on many tumors. In our recent in vitro study, we constructed and evaluated a plasmid for simultaneous silencing of these two targets. In the current study, our aim was to explore the therapeutic potential of gene electrotransfer-mediated delivery of this new plasmid in vivo, and to elucidate the effects of combined therapy with tumor irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice in the syngeneic murine mammary adenocarcinoma tumor model TS/A. Histological analysis of tumors (vascularization, proliferation, hypoxia, necrosis, apoptosis and infiltration of immune cells) was performed to evaluate the therapeutic mechanisms. Additionally, potential activation of the immune response was evaluated by determining the induction of DNA sensor STING and selected pro-inflammatory cytokines using qRT-PCR. The results point to a significant radiosensitization and a good therapeutic potential of this gene therapy approach in an otherwise radioresistant and immunologically cold TS/A tumor model, making it a promising novel treatment modality for a wide range of tumors.