Cell Proliferation In Response Research Articles

Biomedical Applications of Cold Atmospheric Plasma: Cancer Therapy and Virus Inactivation

Cold atmospheric plasma (CAP), known for its ability to generate Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS), has emerged as a promising tool in many biomedical applications, including cancer therapeutics and virus inactivation. In cancer therapy, ROS and RNS induced by plasma have been shown to selectively target cancer cells, triggering apoptosis and inhibiting tumor growth while sparing healthy cells. This selective cytotoxicity makes plasma an attractive option for localized cancer treatment with minimal side effects. Additionally, the antimicrobial properties of ROS and RNS make plasma an effective agent for virus inactivation. By connecting the dots between cancer therapy and virus disinfection, this review paper sheds light on the potential of plasma-generated ROS and RNS as versatile tools in medical applications, offering a dual benefit of targeted cancer treatment and effective virus control. Specifically, this review explores the effects of CAP treatment on B16 melanoma cells, assessing changes in cell viability, proliferation, apoptosis and oxidative stress response. Dichloroacetate (DCA), a mitochondrial modulator, has shown promise in inducing apoptosis in melanoma cells. However, its clinical use is limited by side effects related to its neurotoxicity and suspected carcinogenicity. The potential of CAP in enhancing the therapeutic efficacy of DCA on B16-F10 melanoma apoptosis is also explored. CAP therapeutics for other types of cancers are also explored in this review. Alongside its implications in oncology, CAP has gathered substantial attention for its extraordinary potential in neutralizing viruses. CAP’s exceptional precision in targeting viral envelopes, genetic material and structural components disrupts their integrity and vital functions. This orchestrated mechanism offers a swift and efficacious route to render viruses inactive, encompassing both enveloped and nonenveloped viral types. In parallel pursuit, this review examines CAP-generated reactive species’ proficiency in deactivating SARS-CoV-2 across various surfaces. The inquiry entails subjecting surfaces to CAP treatment, succeeded by quantifying viral load and scrutinizing viral integrity. The outcomes gleaned from these investigations provide pivotal insights into CAP’s potential as a robust surface disinfection strategy, poised to combat the propagation of viral transmission.

Trem2 Agonist Reprograms Foamy Macrophages to Promote Atherosclerotic Plaque Stability.

Trem2 (triggering receptor on myeloid cells 2), a surface lipid receptor, is expressed on foamy macrophages within atherosclerotic lesions and regulates cell survival, proliferation, and anti-inflammatory responses. Studies examining the role of Trem2 in atherosclerosis have shown that deletion of Trem2 leads to impaired foamy macrophage lipid uptake, proliferation, survival, and cholesterol efflux. Thus, we tested the hypothesis that administration of a Trem2 agonist antibody (AL002a) to atherogenic mice would enhance macrophage survival and decrease necrotic core formation to improve plaque stability. To model a therapeutic intervention approach, atherosclerosis-prone mice (Ldlr [low-density lipoprotein receptor]-/-) were fed a high-fat diet for 8 weeks, then transitioned to treatment with AL002a or isotype control for an additional 8 weeks while continuing on a high-fat diet. AL002a-treated mice had increased lesion size in both the aortic root and whole mount aorta, which correlated with an expansion of plaque macrophage area. This expansion was due to increased macrophage survival and proliferation in plaques. Importantly, plaques from AL002a-treated mice showed improved features of plaque stability, including smaller necrotic cores, increased fibrous caps, and greater collagen deposition. Single-cell RNA sequencing of whole aorta suspensions from isotype- and AL002a-treated atherosclerotic mice revealed that Trem2 agonism dramatically altered foamy macrophage transcriptome. This included upregulation of oxidative phosphorylation and increased expression of collagen genes. In vitro studies validated that Trem2 agonism with AL002a promoted foamy macrophage oxidized low-density lipoprotein uptake, survival, and cholesterol efflux. Trem2 agonism expands atherosclerotic plaque macrophages by promoting cell survival and proliferation but improves features of plaque stability by rewiring foamy macrophage function to enhance cholesterol efflux and collagen deposition.

Identification of PDZD11 as a Potential Biomarker Associated with Immune Infiltration for Diagnosis and Prognosis in Epithelial Ovarian Cancer.

Evidence has indicated that PDZD11 is involved in regulating adherens junction. However, the distinct effect of its aberrant expression on epithelial ovarian cancer (EOC) awaits clarification. In this study, public databases (Gene Expression Omnibus, The Cancer Genome Atlas, and The Genotype-Tissue Expression), online analysis tools (Kaplan-Meier plotter and TIMER), and data analysis methods (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and the CIBERSORT algorithm) were fully utilized to analyze the differential expression, diagnostic efficiency, prognostic significance, potential function, and correlation with immune infiltration of PDZD11. The differential expression of PDZD11 was tested by immunohistochemistry in EOC tissues (78 cases) and control tissues (37 cases). Our results indicate that PDZD11 was remarkably overexpressed in EOC, which was associated with advanced cancer stages, no lymphatic metastasis status, and poor prognosis. Moreover, PDZD11 played a role in cell adhesion, cell proliferation, and immune responses. Also, PDZD11 was significantly related to the abundances of infiltrating immune cells in EOC, including neutrophils, macrophages, dendritic cells, CD8+ T cells, and CD4+ T cells, and its expression was positively co-expressed with well-known immune checkpoints, including TIGIT, TIM3, LAG3, CTLA4, and PD-1. These results suggest that PDZD11 could be a potential diagnostic and prognostic biomarker associated with immune infiltration in EOC, and our findings might help elucidate the function of PDZD11 in carcinogenesis.

Spatiotemporal ATF3 Expression Determines VSMC Fate in Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy, likely due to the limited understanding of the mechanisms underlying AAA development and progression. Activating transcription factor (ATF) 3 has been increasingly recognized as a key regulator of cardiovascular diseases. However, the role of ATF3 (activating transcription factor 3) in AAA development and progression remains elusive. Genome-wide RNA sequencing analysis was performed on the aorta isolated from saline or Ang II (angiotensin II)-induced AAA mice, and ATF3 was identified as the potential key gene for AAA development. To examine the role of ATF3 in AAA development, vascular smooth muscle cell-specific ATF3 knockdown or overexpressed mice by recombinant adenoassociated virus serotype 9 vectors carrying ATF3, or shRNA-ATF3 with SM22α (smooth muscle protein 22-α) promoter were used in Ang II (angiotensin II)-induced AAA mice. In human and murine vascular smooth muscle cells, gain or loss of function experiments were performed to investigate the role of ATF3 in vascular smooth muscle cell proliferation and apoptosis. In both Ang II-induced AAA mice and patients with AAA, the expression of ATF3 was reduced in aneurysm tissues but increased in aortic lesion tissues. The deficiency of ATF3 in vascular smooth muscle cell promoted AAA formation in Ang II-induced AAA mice. PDGFRB (platelet-derived growth factor receptor β) was identified as the target of ATF3, which mediated vascular smooth muscle cell proliferation in response to TNF-alpha (tumor necrosis factor-α) at the early stage of AAA. ATF3 suppressed the mitochondria-dependent apoptosis at the advanced stage by upregulating its direct target BCL2. Our chromatin immunoprecipitation results also demonstrated that the recruitment of NFκB1 and P300/BAF/H3K27ac complex to the ATF3 promoter induces ATF3 transcription via enhancer activation. NFKB1 inhibitor (andrographolide) inhibits the expression of ATF3 by blocking the recruiters NFKB1 and ATF3-enhancer to the ATF3-promoter region, ultimately leading to AAA development. Our results demonstrate a previously unrecognized role of ATF3 in AAA development and progression, and ATF3 may serve as a novel therapeutic and prognostic marker for AAA.

Mitochondrial dynamics, quality control and mtDNA in Alcohol-associated liver disease and liver cancer.

Mitochondria are intracellular organelles responsible for energy production, glucose and lipid metabolism, cell death, cell proliferation, and innate immune response. Mitochondria are highly dynamic organelles that constantly undergo fission, fusion, and intracellular trafficking, as well as degradation and biogenesis. Mitochondrial dysfunction has been implicated in a variety of chronic liver diseases including alcohol-associated liver disease (ALD), metabolic dysfunction-associated steatohepatitis (MASH), and hepatocellular carcinoma (HCC). In this review, we provide a detailed overview of mitochondrial dynamics, mitophagy, and mtDNA-mediated innate immune response, and how dysregulation of these mitochondrial processes affects the pathogenesis of ALD and HCC. Mitochondrial dynamics and mtDNA-mediated innate immune response may thereby represent an attractive therapeutic target for ameliorating ALD and alcohol-associated HCC.

Identification of Novel STAT3 Dimerization Inhibitor Through Structure-Based Virtual Screening for Cancer Management

Background: Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that controls cell proliferation, differentiation, angiogenesis, and immunological responses. In many human malignancies, abnormal STAT3 activation promotes tumor growth via oncogenic gene expression, resulting in tumor malignancy. Many drugs with clinically authorized analogues that are used as STAT3 inhibitors for cancer therapy have several drawbacks in terms of stability and toxicity.Methods: This study used PyRx 0.8 tool to screen the Traditional Chinese Medicine (TCM) database of about 32,364 commercially available natural compounds in order to identify new STAT3 inhibitors. Physicochemical and ADME properties of selected compounds were estimated using Datawarrior and SwissADME.Result: The top 20 compounds were initially chosen based on their strong binding affinities with STAT3. Lipinski and Vaber tools were used to filter the top 20 compounds, yielding the top 6 compounds. The compounds ZINC85542844, ZINC4098720, ZINC85543599, ZINC85593523, ZINC85593528, and ZINC85593537 were passed through these filters. These compounds were found to interact with active site STAT3 residues and have several amino acid interactions in common with the control compound (STX0119).Conclusion: This study suggests that the compounds ZINC85542844, ZINC4098720, ZINC85543599, ZINC85593523, ZINC85593528, and ZINC85593537 could be used as a lead for the development of novel STAT3 inhibitors. However, further experimental validation is required to optimized them as STAT3 inhibitors.Keywords: STAT3; Transcription factor; Natural compounds; Cancer; Virtual screening

Adult aberrant astrocytes submitted to late passage cultivation lost differentiation markers and decreased their pro-inflammatory profile

In amyotrophic lateral sclerosis (ALS), astrocytes are considered key players in some non-cell non-neuronal autonomous mechanisms that underlie motor neuron death. However, it is unknown how much of these deleterious features were permanently acquired. To assess this point, we evaluated if the most remarkable features of neurotoxic aberrant glial phenotypes (AbAs) isolated from paralytic rats of the ALS model G93A Cu/Zn superoxide dismutase 1 (SOD1) could remain upon long lasting cultivation. Real time PCR, immunolabelling and zymography analysis showed that upon many passages, AbAs preserved the cell proliferation capacity, mitochondrial function and response to different compounds that inhibit some key astrocyte functions but decreased the expression of parameters associated to cell lineage, homeostasis and inflammation. As these results are contrary to the sustained inflammatory status observed along disease progression in SOD1G93A rats, we propose that the most AbAs remarkable features related to homeostasis and neurotoxicity were not permanently acquired and might depend on the signaling coming from the injuring microenvironment present in the degenerating spinal cord of terminal rats.

Exploring the association of POSTN+ cancer-associated fibroblasts with triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis. Cancer-associated fibroblasts (CAFs) play a critical role in regulating TNBC tumor development. This study aimed to identify and characterize a specific subtype of CAFs associated with TNBC. Initially, using high-throughput bulk transcriptomic data in two cohorts, we identified three CAF-related subtypes (CS1, CS2, CS3) in TNBC samples. These three CAFs subtypes were closely linked to the tumor microenvironment. The CS1 subtype exhibited a relatively immune-rich microenvironment and a favourable prognosis, whereas the CS3 subtype displayed an immune-deprived tumor microenvironment and an unfavourable prognosis. Through WGCNA analysis, POSTN was identified as a key biomarker for CAFs associated with TNBC. Then, POSTN+CAFs was identified and characterized. Both POSTN and POSTN+CAFs showed significant positive correlations with stromal molecules HGF and MET at both the transcriptional and protein levels. Specifically co-localized with CAFs in the tumor stromal area, POSTN, produced by POSTN+CAFs, could modulate the HGF-MET axis, serving as a bypass activation pathway to regulate tumor cell proliferation in response to EGFR inhibitor and MET inhibitor. This study underscores the significance of POSTN and POSTN+CAFs as crucial targets for the diagnosis and treatment of TNBC.

Dysregulation of miR-25-3p in Diabetic Nephropathy and Its Role in Inflammatory Response.

To investigate the expression level of miR-25-3p in patients with type 2diabetes mellitus (T2DM) and diabetic nephropathy (DN), and its effect on proliferation, apoptosis and inflammatory response of mesangial cells cultured with high glucose. Blood samples of all clinical subjects were collected for RT-qPCR analysis to detect serum miR-25-3p levels. Human mesangial cells (HMCs) cultured with high glucose were used to construct DN model in vitro. MTT assay, flow cytometry and ELISA were used to evaluate the effects of miR-25-3p on the proliferation, apoptosis, and inflammatory response of DN cell models. Serum miR-25-3p was decreased in both T2DM group and DN group, but more in DN group. Serum miR-25-3p was positively correlated with eGFR and negatively correlated with UAER. The expression of miR-25-3p was reduced in HMCs induced by high glucose. Transfection of miR-25-3p mimic could significantly up-regulate the miR-25-3p level in HMCs. Besides, high glucose culture resulted in abnormal proliferation of HMCs, reduced apoptotic cells, and increased inflammation. The addition of miR-25-3p mimic significantly inhibited cell proliferation and promoted cell apoptosis and reduced the production of inflammatory factors. The abnormal reduction of serum miR-25-3p in DN indicates that it may be a potential biomarker for clinical diagnosis of DN. In in vitro experiments, miR-25-3p was involved in the progression of DN by regulating cell proliferation, apoptosis, and inflammatory response.

Progress in cancer research on the regulator of phagocytosis CD47, which determines the fate of tumor cells (Review).

Cluster of differentiation 47 (CD47) is a transmembrane protein that is widely and moderately expressed on the surface of various cells and can have an essential role in mediating cell proliferation, migration, phagocytosis, apoptosis, immune homeostasis and other related responses by binding to its ligands, integrins, thrombospondin-1 and signal regulatory protein α. The poor prognosis of cancer patients is closely associated with high expression of CD47 in glioblastoma, ovarian cancer, breast cancer, bladder cancer, colon cancer and hepatocellular carcinoma. Upregulation of CD47 expression facilitates the growth of numerous types of tumor cells, while downregulation of its expression promotes phagocytosis of tumor cells by macrophages, thereby limiting tumor growth. In addition, blocking CD47 activates the cyclic GMP-AMP (cGAMP) synthase/cGAMP/interferon gene stimulating factor signaling pathway and initiates an adaptive immune response that kills tumor cells. The present review describes the structure, function and interactions of CD47 with its ligands, as well as its regulation of phagocytosis and tumor cell fate. It summarizes the therapeutics, mechanisms of action, research advances and challenges of targeting CD47. In addition, this paper provides an overview of the latest therapeutic options for targeting CD47, such as chimeric antigen receptor (CAR) T-cells, CAR macrophages and nanotechnology-based delivery systems, which are essential for future clinical research on targeting CD47.

Abstract CT079: The Pseudovax trial: A novel peptide vaccine targeting mutated GNAS for pseudomyxoma peritonei

Abstract Background: Pseudomyxoma peritonei (PMP) is a rare cancer originating from appendiceal mucinous tumors, which may spread to encompass the entire peritoneal cavity. Complete surgical removal and hyperthermic intraperitoneal chemotherapy cures approximately half of the patients, but patients with recurrent or non-resectable disease have no efficacious treatment options. These patients have a high unmet need for novel treatment options, and in the Pseudovax trial we will explore a novel therapeutic concept. A main molecular driver of PMP (in up to 90% of cases) is otherwise rare mutations in the GNAS oncogene, coding for the Gsα protein. We hypothesized that mutated Gsα would be a tumor neoantigen, and in T cells isolated from peripheral blood of PMP patients, a strong T cell proliferative response was observed. In the corresponding tumors, T cells with up-regulated immune checkpoint molecules PD-1 and TIGIT were detected. Study hypotheses: Patients with GNAS-mutated PMP has a preexisting, attenuated immune response directed against mutated Gsα protein. Vaccination with mutated Gsα peptides can be combined with immune checkpoint inhibition (ICI) with acceptable toxicity. The vaccine will reactivate the immune response and ICI will restore measurable anticancer immunity in patients. Study design: The Pseudovax trial is a small (10 patients) first-in-human, proof-of-concept, prospective, open label, phase I trial. Patients with recurrent or non-resectable PMP are eligible for inclusion. During a treatment period of 12 months, a mutated Gsα peptide vaccine + adjuvant (GM-CSF) will be administered as intradermal injections. After 12 weeks, treatment with ICI will be initiated. The primary endpoints are toxicity and circulating immune response against the vaccine, measured as increase in interferon-gamma production or proliferation of circulating vaccine-specific T cells compared to pre-vaccination baseline. Secondary endpoints are progression-free survival (abdominopelvic CT scans, blood levels of carcinoembryonic antigen (CEA) and/or detection of GNAS mutation by ctDNA analysis). Citation Format: Kjersti Flatmark, Annette Torgunrud, Fleten G. Karianne, Ben Davidson, Hedvig V. Juel, Nadia Mensali, Christin Lund Andersen, Else Marit Inderberg. The Pseudovax trial: A novel peptide vaccine targeting mutated GNAS for pseudomyxoma peritonei [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT079.

Immune system-related plasma extracellular vesicles in healthy aging.

To identify age-related plasma extracellular vehicle (EVs) phenotypes in healthy adults. EV proteomics by high-resolution mass spectrometry to evaluate EV protein stability and discover age-associated EV proteins (n=4 with 4 serial freeze-thaws each); validation by high-resolution flow cytometry and EV cytokine quantification by multiplex ELISA (n=28 healthy donors, aged 18-83 years); quantification of WI-38 fibroblast cell proliferation response to co-culture with PKH67-labeled young and old plasma EVs. The EV samples from these plasma specimens were previously characterized for bilayer structure, intra-vesicle mitochondria and cytokines, and hematopoietic cell-related surface markers. Compared with matched exo-EVs (EV-depleted supernatants), endo-EVs (EV-associated) had higher mean TNF-α and IL-27, lower mean IL-6, IL-11, IFN-γ, and IL-17A/F, and similar mean IL-1β, IL-21, and IL-22 concentrations. Some endo-EV and exo-EV cytokine concentrations were correlated, including TNF-α, IL-27, IL-6, IL-1β, and IFN-γ, but not IL-11, IL-17A/F, IL-21 or IL-22. Endo-EV IFN-γ and exo-EV IL-17A/F and IL-21 declined with age. By proteomics and confirmed by flow cytometry, we identified age-associated decline of fibrinogen (FGA, FGB and FGG) in EVs. Age-related EV proteins indicated predominant origins in the liver and innate immune system. WI-38 cells (>95%) internalized similar amounts of young and old plasma EVs, but cells that internalized PKH67-EVs, particularly young EVs, underwent significantly greater cell proliferation. Endo-EV and exo-EV cytokines function as different biomarkers. The observed healthy aging EV phenotype reflected a downregulation of EV fibrinogen subpopulations consistent with the absence of a pro-coagulant and pro-inflammatory condition common with age-related disease.

Interaction of SMAC with a survivin-derived peptide alters essential cancer hallmarks: Tumor growth, inflammation, and immunosuppression

SMAC/Diablo is known as a pro-apoptotic mitochondrial protein released into the cytosol in response to apoptotic signals. We recently reported SMAC overexpression in cancers as essential for cell proliferation and tumor growth due to non-apoptotic functions including phospholipid synthesis regulation. These functions may be associated with its interactions with partner proteins. Using a peptide array with 768 peptides derived from 11 selected SMAC-interacting proteins, we identified SMAC-interacting sequences. These SMAC-binding sequences were produced as cell penetrating peptides targeted to the cytosol, mitochondria, or nucleus, inhibiting cell proliferation and inducing apoptosis in several cell lines. For in-vivo study, a survivin/BIRC5-derived peptide was selected due to its overexpression in many cancers, and involvement in mitosis, apoptosis, autophagy, cell proliferation, inflammation, and immune responses, as a target for cancer therapy. Specifically, a SMAC-targeting survivin/BIRC5-derived peptide, given intratumorally or intravenously, strongly inhibited lung tumor growth, cell proliferation, angiogenesis, and inflammation, induced apoptosis, and remodeled the tumor microenvironment. The peptide promoted tumor infiltration of CD8+ cells and increased cell-intrinsic PD-1/PD-L1 expression, resulting in cancer cell self-destruction and increased tumor cell death, preserving immune cells. Thus, targeting the interaction between the multifunctional proteins SMAC and survivin represents a innovative therapeutic cancer paradigm.

Cell cycle associated protein 1 associates with immune infiltration and ferroptosis in gastrointestinal cancer

BackgroundCell Cycle-Associated Protein 1 (CAPRIN1) play an important role in cell proliferation, oxidative stress, and inflammatory response. Nonetheless, its role in tumor immunity and ferroptosis is largely unknown in gastrointestinal cancer patients. MethodsThrough comprehensive bioinformatics, we investigate CAPRIN1 expression patterns and its role in diagnosis, functional signaling pathways, tumor immune infiltration and ferroptosis of different gastrointestinal cancer subtypes. Besides, immunohistochemistry (IHC) and immune blot were used to validate our esophagus cancer clinical data. The ferroptotic features of CAPRIN1 in vitro were assessed through knockdown assays in esophagus cancer cells. ResultsCAPRIN1 expression was significantly upregulated, correlated with poor prognosis, and served as an independent risk factor for most gastrointestinal cancer. Moreover, CAPRIN1 overexpression positively correlated with gene markers of most infiltrating immune cells, and immune checkpoints. CAPRIN1 knockdown significantly decreased the protein level of major histocompatibility complex class I molecules. We also identified a link between CAPRIN1 and ferroptosis-related genes in gastrointestinal cancer. Knockdown of CAPRIN1 significantly increased the production of lipid reactive oxygen species and malondialdehyde. Inhibition of CAPRIN1 expression promoted ferroptotic cell death induced by RAS-selective lethal 3 and erastin in human esophagus cancer cells. ConclusionCollectively, our results demonstrate that CAPRIN1 is aberrantly expressed in gastrointestinal cancer, is associated with poor prognosis, and could potentially influence immune infiltration and ferroptosis.

TRIM52 knockdown inhibits proliferation, inflammatory responses and oxidative stress in IL-1β-induced synovial fibroblasts to alleviate temporomandibular joint osteoarthritis.

To explore the mechanism of tripartite motif 52 (TRIM52) in the progression of temporomandibular joint osteoarthritis (TMJOA). Gene and protein expression were tested by quantitative real-time polymerase chain reaction and western blot, respectively. The levels of pro-inflammatory cytokines and oxidative stress factors were evaluated using enzyme-linked immunosorbent assay and biochemical kit, respectively. Cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays were carried out to assess cell proliferation. Immunofluorescence was used to detect the expression of CD68 and Vimentin in primary synovial fibroblasts (SFs). Haematoxylin and eosin staining and Safranin O/Fast green were used to evaluate the pathological damage of synovial and cartilage tissue in rats. TRIM52 was upregulated in the synovial tissue and SFs in patients with TMJOA. Interleukin (IL)-1β treatment upregulated TRIM52 expression in TMJOA SFs and normal SF (NSF), promoting cell proliferation, inflammatory response and oxidative stress in NSF, SFs. Silence of TRIM52 relieved the cell proliferation, inflammatory response and oxidative stress induced by IL-1β in SFs, while overexpression of TRIM52 enhanced IL-1β induction. Meanwhile, IL-1β induction activated toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB pathway, which was augmented by upregulation of TRIM52 in NSF, and was attenuated by TRIM52 knockdown in SFs. Besides, pyrrolidinedithiocarbamic acid ameliorated IL-1β-induced proliferation and inflammatory response by inhibiting TLR4/NF-κB signalling. Meanwhile, TRIM52 knockdown inhibited cell proliferation, oxidative stress and inflammatory response in IL-1β-induced SFs through downregulation of TLR4. TRIM52 promoted cell proliferation, inflammatory response, and oxidative stress in IL-1β-induced SFs. The above functions were mediated by the activation of TLR4/NF- κB signal pathway.

Abstract 1631: A homogeneous assay system for discernment of proliferative, antiproliferative, and cytotoxic effects in culture

Abstract We describe a novel homogeneous cell-based assay developed for the quantitative detection of Ki-67 protein, a recognized marker of cell proliferation, utilizing Lumit™ immunoassay technology. This luminescent assay has been effectively multiplexed with a fluorescent measure of cell death, employing a cell-impermeant fluorogenic DNA dye. This multiplex allows for the clear distinction between antiproliferative activity and overt cytotoxicity in cell culture models. Through varying lytic conditions, we demonstrated effective cell lysis and detection of nuclear Ki-67 protein. Specifically, Lumit™ Lysis Buffer II provided efficient recovery of Ki-67 protein without compromising assay performance, including the requisite binding of labeled antibodies to the target protein. This assay exhibited excellent signal-to-background (up to 40-fold) and signal linearity (R^2 = 0.99) in wildtype HeLa cells (~1000 to 80,000/well), while the signal in Ki-67 knockout HeLa cells was comparable to the no-cell background, indicating excellent specificity for Ki-67 protein. Due to the unavailability of recombinant full-length Ki-67 protein, two different recombinant Ki-67 protein fragments were used to corroborate assay sensitivity of <1 pM Ki-67 protein. The assay's suitability for human Ki-67 protein detection, not mouse, was observed through further cell-based testing. The proliferation/cytotoxicity assay multiplex was validated in both suspension and adherent cell lines, as well as primary cell culture, detecting antiproliferative and proliferative responses. CDK4/6 inhibitors are a class of antiproliferative agents of significant interest for their potential in treating cancer. In Jurkat cells, treatment with the CDK4/6 inhibitor palbociclib led to a dose-dependent reduction in Ki-67 protein expression with an EC50 of 88 nM, and total Ki-67 elimination at 2 μM compound concentration. Notably, there was no cytotoxicity detected at the tested doses and treatment duration (up to 48 h), but a significant reduction in viable cell number at maximal palbociclib concentrations, aligning with an antiproliferative mechanism. This work establishes a rapid, simple, luminescent assay for determining Ki-67 levels that can be paired with a fluorogenic readout of cell death, facilitating efficient and quantitative discernment of altered proliferation in mammalian cell culture using standard microplate readers. Citation Format: Dan F. Lazar, Kevin R. Kupcho, Andrew L. Niles, James J. Cali. A homogeneous assay system for discernment of proliferative, antiproliferative, and cytotoxic effects in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1631.

Omentin-1 inhibits the development of benign prostatic hyperplasia by attenuating local inflammation

BackgroundBenign prostatic hyperplasia (BPH) is a prevalent disease affecting elderly men, with chronic inflammation being a critical factor in its development. Omentin-1, also known as intelectin-1 (ITLN-1), is an anti-inflammatory protein primarily found in the epithelial cells of the small intestine. This study aimed to investigate the potential of ITLN-1 in mitigating BPH by modulating local inflammation in the prostate gland.MethodsOur investigation involved two in vivo experimental models. Firstly, ITLN-1 knockout mice (Itln-1−/−) were used to study the absence of ITLN-1 in BPH development. Secondly, a testosterone propionate (TP)-induced BPH mouse model was treated with an ITLN-1 overexpressing adenovirus. We assessed BPH severity using prostate weight index and histological analysis, including H&E staining, immunohistochemistry, and enzyme-linked immunosorbent assay. In vitro, the impact of ITLN-1 on BPH-1 cell proliferation and inflammatory response was evaluated using cell proliferation assays and enzyme-linked immunosorbent assay.ResultsIn vivo, Itln-1−/− mice exhibited elevated prostate weight index, enlarged lumen area, and higher TNF-α levels compared to wild-type littermates. In contrast, ITLN-1 overexpression in TP-induced BPH mice resulted in reduced prostate weight index, lumen area, and TNF-α levels. In vitro studies indicated that ITLN-1 suppressed the proliferation of prostate epithelial cells and reduced TNF-α production in macrophages, suggesting a mechanism involving the inhibition of macrophage-mediated inflammation.ConclusionThe study demonstrates that ITLN-1 plays a significant role in inhibiting the development of BPH by reducing local inflammation in the prostate gland. These findings highlight the potential of ITLN-1 as a therapeutic target in the management of BPH.

Abstract 1644: Exploring the role of a TERT intronic tandem repeat in regulating cell proliferative responses to environmental factors and cancer risk

Abstract The chr 5p15.33 genomic locus encoding telomerase reverse transcriptase (TERT) is structurally complex, with several highly polymorphic intronic variable number tandem repeats (VNTRs). A germline VNTR located in TERT intron 6 (VNTR6-1, 38 bp repeat unit) has previously been associated with TERT alternative splicing but its potential role in regulating cellular functions and disease risk is unclear. To functionally characterize VNTR6-1, we deleted this region (1-2 kb) using CRISPR/Cas9 in UMUC3 (bladder cancer cell line). Cell count growth tracking of parental and VNTR6-1 knockout (VNTR6-1 KO) cells via automated live-cell monitoring revealed different relative growth rates under serum starvation vs. normal conditions, suggesting that VNTR6-1 might be sensitive to growth factors present in culture media. Evaluation of growth in media with full vs. charcoal-stripped (CS) serum, showed significant differences, with higher cell counts in parental vs. VNTR6-1 KO in CS-serum (P=4.1e-3), parental vs. parental in full (P=1.57e-4) or CS-serum (P=4.76e-2), but not in parental vs. VNTR6-1 KO in full serum (P=0.378). After 5-6 days of growth, cells in CS-serum also rapidly began outgrowing cells in full serum. We observed that VNTR6-1 KO dampened the outgrowth response in cells lacking growth factors in CS-serum. Proliferation-specific growth assays tracking the dilution of an incorporated dye in cells by flow cytometry further showed that VNTR6-1 KO leads to more pronounced proliferation differences due to the presence or absence of serum-supplied factors. Overall, cell growth patterns appear to be sensitive to serum in interaction with VNTR6-1, suggesting a potential VNTR6-1-dependent response to extracellular signaling. In human populations, we have identified two VNTR6-1 groups: Short (24-27 copies) and Long (40.5-66.5 copies). In the PLCO dataset (n=100,445 cancer cases and controls), logistic regression showed that VNTR6-1-Long group was associated with a decreased risk in male-prevalent cancers such as bladder and prostate, but an increased risk in female-prevalent cancers such as breast, endometrial and ovarian. These results support a link between the TERT region, and potentially VNTR6-1, with hormone signaling and cancer risk. Motif analysis within VNTR6-1 predicted binding sites for several transcription factors, including those associated with development and cell growth. Together, these results suggest that VNTR6-1 may be involved in regulating cell growth, potentially by facilitating responses to extracellular signaling pathways. Additionally, given the location of VNTR6-1 within a multi-cancer GWAS region, our findings provide functional context for the role of this polymorphic repeat in differential risk and outcomes in certain cancers. Citation Format: Michelle Ho, Oscar Florez-Vargas, Maxwell Hogshead, Brenen Papenberg, Chia Han Lee, Chi Zhang, Kaitlin Forsythe, Wusheng Yan, Raj Chari, Ludmila Prokunina-Olsson. Exploring the role of a TERT intronic tandem repeat in regulating cell proliferative responses to environmental factors and cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1644.

The hTERT and iCasp9 Transgenes Affect EOMES and T-BET Levels in NK Cells and the Introduction of Both Genes Improves NK Cell Proliferation in Response to IL2 and IL15 Stimulation.

The NK cell exhaustion state evolving during extensive and prolonged cultivation is still one of the limitations of NK cell approaches. In this research, we transduced NK cells with the hTERT and iCasp9 genes. hTERT overexpression can prevent the functional exhaustion of NK cells during long-term cultivation, but, still, the therapeutic use of such cells is unsafe without irradiation. To overcome this obstacle, we additionally transduced NK cells with the iCasp9 transgene that enables the rapid elimination of modified cells. We compared the proliferative and functional activities of the hTERT- and/or iCasp9-modified NK cells, determined their exhaustion state and monitored the levels of EOMES and T-BET, the main NK cell transcription factors. The hTERT and iCasp9 genes were shown to affect the EOMES and T-BET levels differently in the NK cells. The EOMES+T-BET+ phenotype characterized the functionally active NK cells during two months of culture upon stimulation with IL2 and K562-mbIL21 feeder cells, which induced the greatest expansion rates of the NK cells, independently of the transgene type. On the other hand, under cytokine stimulation, the hTERT-iCasp9-NK cells displayed improved proliferation over NK cells modified with iCasp9 alone and showed an increased proliferation rate compared to the untransduced NK cells under stimulation with IL2 and IL15, which was accompanied by reduced immune checkpoint molecule expression. The individual changes in the EOMES and T-BET levels strictly corresponded to the NK cell functional activity, the surface levels of activating and inhibitory receptors along with the expansion rate and expression levels of pro-survival and pro-apoptotic genes.

Ascertaining sensitive exposure biomarkers of various metal(loid)s to embryo implantation

Close relationships exist between metal(loid)s exposure and embryo implantation failure (EIF) from animal and epidemiological studies. However, there are still inconsistent results and lacking of sensitive metal(loid) exposure biomarkers associated with EIF risk. We aimed to ascertain sensitive metal(loid) biomarkers to EIF and provide potential biological explanations. Candidate metal(loid) biomarkers were measured in the female hair (FH), female serum (FS), and follicular fluid (FF) with various exposure time periods. An analytical framework was established by integrating epidemiological association results, comprehensive literature searching, and knowledge-based adverse outcome pathway (AOP) networks. The sensitive biomarkers of metal(loid)s along with potential biological pathways to EIF were identified in this framework. Among the concerned 272 candidates, 45 metal(loid)s biomarkers across six time periods and three biomatrix were initially identified by single-metal(loid) analyses. Two biomarkers with counterfactual results according to literature summary results were excluded, and a total of five biomarkers were further determined from 43 remained candidates in mixture models. Finally, four sensitive metal(loid) biomarkers were eventually assessed by overlapping AOP networks information, including Se and Co in FH, and Fe and Zn in FS. AOP networks also identified key GO pathways and proteins involved in regulation of oxygen species biosynthetic, cell proliferation, and inflammatory response. Partial dependence results revealed Fe in FS and Co in FH at their low levels might be potential sensitive exposure levels for EIF. Our study provided a typical framework to screen the crucial metal(loid) biomarkers and ascertain that Se and Co in FH, and Fe and Zn in FS played an important role in embryo implantation.