Enzyme-linked Immunosorbent Assay Research Articles

Abstract 3959: GAS-Luc2 reporter cancer cell lines demonstrating superior performance to the industrial standard interferon-gamma ELISA in 2-D and 3-D ex vivo immune activation and drug screening

Abstract Interferon-gamma (IFN-γ), a cytokine critical for activating cellular immunity and facilitating anti-tumor responses, has become a key cytokine for assessing the efficacy of cancer immunotherapy drugs. While enzyme-linked immunosorbent assay (ELISA) is widely employed for the detection of IFN-γ, it has shown significant limitations in detecting low levels of IFN-γ during early-stage immune activation. Moreover, it presents serious challenges in accurately quantifying paracrine signaling of IFN-γ secreted by immune cells within 3-D co-culture models. To address the rapidly growing demand for more effective monitoring of immune activation for cancer immunotherapy research and better assessment of immunotherapy drug candidates, we developed immune activation reporter cancer cell lines engineered with a gamma-interferon activation site (GAS)-response element positioned upstream of luciferase gene. Upon activation of IFN-γ signaling pathway, these cells express luciferase, allowing for simple detection and quantification to measure immune activation. The cell lines were selected based on a comprehensive protein profiling to ensure endogenous expression of immune checkpoint ligands, such as PD-L1, CD155, or B7-H3, for additional benefit in application in immune checkpoint research. To evaluate the system, the reporter cells were stimulated with varying concentrations of IFN-γ, treated with conditioned media from primary T cells, or co-cultured with IFN-γ-producing primary immune cells. Additionally, the reporter cells were co-cultured in 2-D or 3-D systems for the comparison with ELISA. Our data revealed that bioluminescence intensity from the reporter cells increased by approximately 100- to 250-fold in a dose dependent manner following IFN-γ stimulation. Primary T-cell conditioned media stimulation resulted in a 50- to 100-fold increase in bioluminescence intensity. In co-culture assays with primary T cells or NK cells in the presence of immune checkpoint inhibitors, the reporter cell lines exhibited a 3- to 12-fold increase in bioluminescence intensity. Notably, in both 2-D and 3-D co-culture systems, the reporter cells generated a robust bioluminescent signal even at the IFN-γ concentrations below the detection range of conventional ELISA, highlighting their superior sensitivity and versatility. These reporter cell lines can serve as a valuable standard for early-stage monitoring of immune activation and offer a convenient and highly sensitive platform for evaluating cancer immunotherapy drug candidates. Citation Format: Hyeyoun Chang, John Foulke, Luping Chen, Catherine McManus, Fang Tian, Zhizhan Gu. GAS-Luc2 reporter cancer cell lines demonstrating superior performance to the industrial standard interferon-gamma ELISA in 2-D and 3-D ex vivo immune activation and drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3959.

Abstract 5718: Molecular determinants of platinum resistance in ovarian cancer: Linking PARP1, DNMT1, γ-GCS expression, and LINE1 hypermethylation

Abstract Background: Ovarian cancer is a leading cause of gynecological cancer-related mortality worldwide. Alarmingly, 80% of patients are diagnosed at an advanced stage (FIGO stage III-IV), where the prognosis is poor. The current standard of care for advanced epithelial ovarian cancer (EOC) includes cytoreductive surgery followed by taxane and platinum-based chemotherapy. Despite initial responses, resistance to platinum remains a significant cause of treatment failure and relapse in EOC. This study aims to investigate the molecular underpinnings of platinum resistance by examining the expression and enzymatic activity of PARP1 and γ-GCS and their correlation with DNA methylation patterns in ovarian cancer. Based on existing evidence, we hypothesize that inhibition of PARP1 or γ-GCS may enhance cisplatin (CDDP) sensitivity, offering new therapeutic opportunities. Understanding these mechanisms could pave the way for innovative strategies to overcome chemotherapy resistance in ovarian cancer. Material and Methods: Peripheral Blood (PB) and Ascitic Fluid (AS) samples were collected from 20 EOC patients. The cohort was divided into two groups: Group A (n=10) included treatment-naive baseline patients, and Group B (n=10) comprised relapsed patients. Additionally, healthy control samples (n=10) were collected for comparison. The study involved: 1 Gene Expression Analysis: Differential expression levels of PARP1, DNMT1, and γ-GCS were evaluated using qPCR. 2 Enzymatic Activity Measurement: Glutathione (GSH) activity was quantified using ELISA. 3 Global DNA Methylation Assessment: LINE1 methylation levels were measured via pyrosequencing to examine epigenetic changes. The resulting data were correlated with clinico-pathological parameters, including age, stage, CA markers, and hematological indices. Statistical analysis was conducted using GraphPad Prism 6, and a p-value < 0.05 was considered statistically significant. Results: High γ-GCS expression increases glutathione levels in newly diagnosed ovarian cancer patients, potentially driving platinum resistance. Consistent LINE1 hypermethylation across patients, influenced by various factors, highlights its potential as both a biomarker and a therapeutic target, paving the way for strategies to overcome chemotherapy resistance. Abbreviations: Epithelial Ovarian Cancer (EOC), cisplatin (CDDP), Glutathione (GSH), Gamma Glutamyl Cysteine Synthetase (γ-GCS), Poly (ADP ribosyl) polymerase-1 (PARP1), Enzyme-linked immunosorbent assay (ELISA), Quantitative Polymerase Chain Reaction (qPCR), DNA methylation LINE1, Peripheral Blood (PB), Ascitic Fluid (AS). Citation Format: Luxmi Devi, Ashok Kumar, Lalit Kumar. Molecular determinants of platinum resistance in ovarian cancer: Linking PARP1, DNMT1, γ-GCS expression, and LINE1 hypermethylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5718.

Sero-detection of Hepatitis B Virus Surface Ag among Drugs Upusers in Prisons by Using Enzyme Linked Immunosorbent Assay (ELISA) at Khartoum State

Hepatitis B virus (HBV), a vaccine-preventable infection, remains one of the leading causes of acute and chronic liver diseases. The resulting cirrhosis and hepatocellular carcinoma were the main contributing factors to HBV burden. Hepatitis B is one of the main infectious diseases among people who use drugs (PWUD). There are several reasons why PWUD are considered more vulnerable to HBV. Needle sharing is one of the major routes of transmission among people who inject drugs (PWID). Substance use contributes to other certain vulnerabilities, such as homelessness, incarceration and unsafe sexual contacts. In addition, this population has limited access to health services required for timely prevention, diagnosis, and treatment. The Objective: To determine the prevalence of HBsAg among drug users in prison and assess associated risk factors. Methods and Materials: A case-control study was conducted in Kober and Al Huda prisons, Khartoum State, from May to August 2022. A total of 150 prisoners with a history of drug use were enrolled. Blood samples were collected and analyzed using ELISA to detect HBsAg. Statistical analysis was performed to assess associations between infection status and risk factors such as needle sharing. Results: Among 150 drug users, 30 (20%) were HBsAg positive, while 120 (80%) were negative. The study found a significant association between needle sharing and HBsAg positivity (p = 0.003). The highest prevalence was among heroin users (8.0%) and those aged 30-50 years (13.3%). Males had a higher prevalence (15.3%) than females (4.7%), though this difference was not significant (p = 0.168). Conclusion: HBV prevalence among drug users is considerable. Strengthening harm reduction programs, promoting vaccination, and implementing regular screening in prisons are crucial to reducing HBV transmission.

Exosomes secreted from adipose-derived stem cells inhibit M1 macrophage polarization ameliorate chronic endometritis by regulating SIRT2/NLRP3.

Chronic endometritis (CE) is a key factor in adverse pregnancy outcomes such as miscarriage and infertility. Macrophages are an important immune cell type that secrete pro-inflammatory and anti-inflammatory cytokines that are essential for maintaining endometrial function. This study aimed to investigate the key mechanisms by which exosomes derived from adipose-derived mesenchymal stem cells (ADSCs) regulate macrophage polarization through the sirtuin 2 (SIRT2)/NOD-like receptor pyrin containing 3 (NLRP3) axis and exert a protective effect on CE. Exosomes were obtained from ADSCs (ADSCs-exo) using the classical ultracentrifugation method and characterized using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. ADSCs-exo protective effects on CE mice and RAW 264.7 cells and its related molecular mechanisms were investigated using real-time quantitative polymerase chain reaction, western blotting, enzyme-linked immunosorbent assay, flow cytometry, immunofluorescence, immunoprecipitation, hematoxylin and eosin staining, and immunohistochemistry. ADSCs-exo significantly inhibited M1 macrophage polarization, as evidenced by a 54% reduction in tumor necrosis factor alfa (TNF-α), a 46% reduction in interleukin 1β (IL-1β), and a 36% reduction in interleukin 6 (IL-6) levels in LPS-induced RAW264.7 cells. In vivo, ADSCs-exo treatment reduced the expression of TNF-α by 50%, IL-1β by 58%, and IL-6 by 49% in the uterine tissues of CE mice. Moreover, ADSCs-exo upregulated the expression of SIRT2, promoted the deacetylation modification of NLRP3 to inhibit NLRP3 inflammasome activation, and further suppressed M1 macrophage polarization. However, these trends were reversed after SIRT2 silencing. Our experimental results demonstrate that ADSCs-exo alleviate CE by regulating the SIRT2/NLRP3 axis to inhibit M1 macrophage polarization. This provides a potential theoretical basis for the therapeutic role of stem cells in CE.

In Vitro Analysis of Healing Properties of Platelet-Rich Fibrin (PRF) vs. Collagen Membranes in Guided Tissue Regeneration

ABSTRACT Aim: The present study sought to assess, using growth factor release and tissue integration, the in vitro healing characteristics of collagen membranes and platelet-rich fibrin (PRF) in guided tissue regeneration (GTR). Materials and Methods: The control was commercially available collagen membranes; PRF membranes were produced using a normal centrifugation technique. Scanning electron microscopy (SEM) was used to examine structural features. Key growth factors—including transforming growth factor-beta 1 (TGF-β1), platelet-derived growth factor (PDGF-BB), and vascular endothelial growth factor (VEGF)—were assessed using enzyme-linked immunosorbent assays (ELISA). Using scratch tests, we assessed wound healing capacity and fibroblast migration. The student’s t-test was used for statistical analysis; P < 0.001 was considered to be significant. Results: While collagen membranes showed a consistent porosity shape, PRF showed a rich fibrin network, including embedded platelets and leukocytes. At 14 days, PRF showed much more cell growth (P < 0.001). Growth factor analysis showed that PRF generated more TGF-β1, PDGF-BB, and VEGF with consistent release (P < 0.001). PRF shows greater healing properties than collagen membranes with enhanced cellular proliferation, movement, and release of growth factors. Conclusion: These results suggest that PRF might be a more effective biomaterial for use in guided tissue regeneration.

Abstract 2639: Role of annexin A2 in triple-negative breast cancer metastasis

Introduction: Emerging evidence indicates that AnxA2 plays a role in the invasion and metastasis of breast cancer. However, the clinical significance of AnxA2 expression in breast cancer remains unknown. Methods/Materials: The TNBC cells were purchased from the American Type Culture Collection (Manassas, VA). The expression of AnxA2 in cell lines, tumor tissues, and serum samples of breast cancer patients were analyzed by immunoblotting, immunohistochemistry, and enzyme-linked immunosorbent assay, respectively. Results: We observed that AnxA2 was significantly elevated in the tumor tissues and serum samples of breast cancer patients compared to normal controls. High levels of serum AnxA2 were significantly correlated with tumor grade and poor survival in breast cancer patients. When we analyzed molecular subtypes, we found that AnxA2 expression was notably higher in the tumor tissues and serum samples of triple-negative breast cancer (TNBC) patients compared to those with other breast cancer subtypes. Our studies on breast cancer cell lines indicated that the secretion of AnxA2 is linked to its phosphorylation at tyrosine 23 (Tyr23). Additionally, phosphorylation of AnxA2 at Tyr23 facilitates its translocation to the cell surface in TNBC cell lines. The increased expression of AnxA2 on the cell surface enhances plasmin generation in these cell lines, and an inhibitory hexapeptide against AnxA2 can neutralize this activity. Furthermore, our analysis of AnxA2 phosphorylation in clinical samples confirmed that the phosphorylation at Tyr23 was significantly higher in the tumor tissues of TNBC patients when compared to matched adjacent non-tumorigenic breast tissues. Conclusions: These findings suggest that serum AnxA2 concentration could be a potential diagnostic biomarker for TNBC patients. Citation Format: Pankaj Chaudhary. Role of annexin A2 in triple-negative breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2639.

Abstract 1665: A comprehensive platform for the screening and assessment of IRAK4-targeting PROTACs

Abstract Interleukin-1 receptor-associated kinase 4 (IRAK4) serves as a pivotal mediator in the signaling cascades of both the interleukin 1 receptor (IL-1R) and toll-like receptor (TLR) pathways. It is crucial for myddosome signaling, as its kinase and scaffolding activities are indispensable for the proper formation and signaling of the myddosome complex. This complex, in turn, initiates the activation of key downstream pathways, including NFkB and interferon-responsive factors 5/7 (IRF 5/7), culminating in the release of inflammatory cytokines and chemokines such as TNF-α, IL-6, IL-1β, and IL-23. These molecules are associated with the development of various autoimmune diseases, such as pyogenic tonsillitis, psoriasis, and atopic dermatitis, positioning IRAK4 as a promising therapeutic target for dampening inflammation mediated by TLR/IL-1R. In this context, we have developed a comprehensive screening and evaluation platform for IRAK4 that spans from in vitro to in vivo studies. The platform encompasses an array of assays at both the biochemical and cellular levels. It features tests for binary and ternary complexes, intracellular detection of ternary complex formation, and a suite of methods for detecting target protein degradation, including flow cytometry, enzyme-linked immunosorbent assay (ELISA), and HiBiT. The platform also offers cellular function assays, such as cytokine release assays utilizing peripheral blood mononuclear cells (PBMCs) and whole blood, as well as assays focused on proteins associated with signal transduction pathways. It also includes off-target and selectivity testing, and extends to in vivo efficacy assessments within the IMQ-induced psoriasis model. Collectively, these assays are designed to facilitate a swift and thorough discovery and evaluation process for IRAK4-targeting PROTACs. Citation Format: Zhaoxia Yin, Xiaojian Wang, Tj (Tiejun) Bing. A comprehensive platform for the screening and assessment of IRAK4-targeting PROTACs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1665.

Abstract 6742: High-affinity human IgG1 antibody against GUCY2C for ADC development: Identification, characterization, and efficacy

Abstract Colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer mortality worldwide. Despite the early disease screening techniques could reduce the number of deaths, the treatments for metastatic CRC still remains limited. GUCY2C, a receptor predominantly found in the intestinal epithelium, is a tumor-associated antigen that is overexpressed in gastrointestinal malignancies, particularly colorectal cancer (CRC). GUCY2C offers a super clean therapeutic target, due to restricted expression in normal tissues and high expression in CRC cells, with significantly reduced the on-target-off-tumor toxicities. A high-affinity, high-specificity human IgG1 monoclonal antibody against GUCY2C was obtained from a 100 billion-capacity human Fab phage display library. This antibody, after recombinant expression in CHO cells, was extensively validated both in vitro and in vivo for its suitability in antibody-drug conjugate (ADC) development. The recombinant antibody demonstrated high affinity for its specific antigen, as confirmed by Bio-Layer Interferometry (BLI) and Enzyme-Linked Immunosorbent Assay (ELISA) methods. Flow cytometry further confirmed that the antibody had picomolar (pM) affinity for gastric and colorectal cancer cell lines expressing high levels of GUCY2C. Cellular internalization experiments indicated that the antibody possesses excellent endocytic capabilities, making it particularly suitable for ADC development. The antibody was then conjugated with a linker-payload complex containing a topoisomerase I inhibitor and an antimetabolite moiety, and subjected to a series of in vitro and in vivo evaluations. The ADC achieved in vitro proliferation inhibition in the picomolar range of IC50. Several in vivo CDX model experiments also demonstrated significant tumor suppression efficacy. Moreover, acute toxicity evaluation of the GUCY2C-ADC in diverse preclinical models indicated favorable safety profiles and tolerability. Overall, these compelling preclinical data supports the promising therapeutic potential of this ADC in targeted treatment of CRC. Citation Format: Yunxia Zheng, Xiafen Wu, Junxiang Jia, Huihui Guo, Xiangfei Kong, Gengxiang Zhao, Yuanyuan Huang, Hangbo Ye, Xiaolei Liu, Yi Luo, Xiaoqiang Xie, Xia Zhou, Lu Bai, Wenjun Li, Binbin Chen, Qingliang Yang, Robert Yongxin Zhao. High-affinity human IgG1 antibody against GUCY2C for ADC development: Identification, characterization, and efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6742.

Serum sirtuin 3 levels and multimodal abnormalities in brain structure and function in parkinson’s disease patients with depression

Abstract Background Depression is a common nonmotor symptom in patients with Parkinson's disease (PD). Currently, few studies have investigated the relationships between serum markers and neuroimaging changes associated with depression in PD patients. Objective To explore the correlations among depression, serum SIRT3 levels, and brain structural and functional alterations in PD patients. Methods The Hamilton Depression Scale‐17 (HAMD-17) was used to assess depression. Serum SIRT3 levels were measured using an enzyme-linked immunosorbent assay (ELISA). Voxel-based morphometry (VBM) and resting-state functional magnetic resonance imaging (rs-fMRI) were used to examine structural and functional alterations. Results Compared to healthy individuals, serum SIRT3 levels were lower in PD patients, especially in those with depression. PD patients with depression had lower total gray matter volume/total intracranial volume (GMV/TIV) ratio, and GMVs of the right amygdala, lower fractional amplitude of low-frequency fluctuations (fALFF) values of the left middle frontal gyrus (MidFG.L) and left superior parietal lobule (SPL.L), and altered functional connectivity(FC) primarily involving the Salience Network (SN) and the default Mode Network (DMN) compared to those without depression. Serum SIRT3 levels, total GMV/TIV ratios, and fALFF values of the MidFG.L and SPL.L have diagnostic value for PD patients with depression, and their combination can improve predictive accuracy. Conclusions Depression in PD patients is associated with lower serum SIRT3 levels, right amygdala atrophy, decreased spontaneous activity in MidFG.L and SPL.L, and altered FC in the DMN and SN.

Abstract 1930: Advancing cervical cancer early detection: A novel lateral flow immunoassay targeting p16INK4a

Cervical cancer ranks as the fourth most prevalent cancer among women globally, with a stark disparity in disease burden and mortality between high-income and low- to middle-income countries (LMICs). Sub-Saharan Africa faces the highest incidence and mortality rates, primarily due to limited access to preventive measures such as vaccines, screening, early detection, and treatment. With approximately 90% of cervical cancer cases occurring in LMICs, there is an urgent need for effective, affordable screening solutions. This study addresses this critical gap by developing a cost-effective, easy-to-use point-of-care (POC) screening test. Specifically, we aim to create a lateral flow immunoassay (LFIA) to detect the overexpression of cyclin-dependent kinase inhibitor 2A (CDKN2A or p16INK4a), a key tumor suppressor protein overexpressed in cervical cancer. The LFIA is designed to be capable of detecting p16INK4a in precancerous and cancerous cells from cervical swab samples, thereby providing an accessible and efficient tool for cervical cancer screening in resource-limited settings. The developed POC LFIA utilizes antigen-antibody interactions. It has four key components: a sample pad, a conjugate pad, a reaction membrane, and an absorbent pad. Cervical swab samples applied to the sample pad rehydrate the dried conjugates, allowing them to interact with the analyte and migrate to the reaction zone, where a gold nanoparticle (AuNP)-conjugated antibody facilitates detection. Initial validation of p16INK4a levels in clinical swab samples was performed using an enzyme-linked immunosorbent assay, which revealed significant overexpression of p16INK4a in high-grade squamous intraepithelial lesions (HSIL), confirming its role as a biomarker for cervical precancerous conditions. Biolayer interferometry was used to select antibodies with optimal nanomolar to picomolar binding affinities. The chosen antibody was conjugated with AuNPs through passive adsorption, achieving a stable 100:1 molar antibody-to-AuNP ratio over seven days. The test and control lines were optimized using in-house dipsticks, with 1.5 mg/mL anti-mouse immunoglobulin G proving ideal for the control line. The current test can reliably detect p16INK4a concentrations as low as 0.5 μg/mL, with further refinements underway to lower the detection limit for clinical use. This simple and cost-effective LFIA offers robust performance, addressing the critical need for affordable and accessible cervical cancer screening, particularly in LMICs. With continued optimization, this tool holds significant promise for improving early detection and treatment outcomes in resource-limited settings. Keywords: point-of-care test, cervical cancer, p16INK4a, early detection, prevention, LMICs Citation Format: Samrin F. Habbani,Sayeh J. Dowlatshahi,Scott C. Bolton,Lucy T. Tecle,Lisa Flowers,Jacqueline C. Linnes,Sulma I. Mohammed. Advancing cervical cancer early detection: A novel lateral flow immunoassay targeting p16INK4a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1930.

Abstract 3318: Serum Insulin-Like Growth Factor-1 levels predict outcomes in patients with advanced hepatocellular carcinoma receiving immunotherapy

Abstract Background: The combination of immune checkpoint inhibitors (ICIs) with antiangiogenesis agents or another ICI has become the standard of care for advanced hepatocellular carcinoma (HCC). Elevated serum levels of insulin-like growth factor-1(IGF-1) levels have been previously associated with better outcomes of patients with advanced HCC treated with sorafenib or atezolizumab plus bevacizumab. However, the prognostic value of serum IGF-1 level for other immunotherapy regimens remains unexplored. Methods: Patients with advanced HCC who received anti-PD-1/anti-PD-L1 monoclonal antibody monotherapy or combinations were enrolled. Serum samples were collected prior to initiating immunotherapy, and serum IGF-1 levels were quantified using enzyme-linked immunosorbent assay (ELISA). Based on prior findings from our group, a cutoff value of 63.6 ng/mL was used to stratify patients into high and low serum IGF-1 groups. Patient demographics, tumor characteristics, treatment responses, and survival outcomes were analyzed. Results: From August 2015 to July 2024, 111 patients were enrolled. Baseline characteristics were as follows: 90% male, 71% HBsAg-positive, 18% anti-HCV-positive, Child-Pugh scores: 72% (5 points)/23% (6 points)/5% (7 points), 43% with macrovascular invasion (MVI), 72% with extrahepatic involvement (EHI), and 46% treated with antiangiogenic-containing regimens. Patients with higher serum IGF-1 levels demonstrated fewer adverse clinical features, including lower Child-Pugh scores, ALBI grades, and MVI rates. Patients who achieved an objective response to therapy had significantly higher IGF-1 levels compared to those who did not (57.2 vs. 46.2 ng/mL, p = 0.037). Additionally, patients with high IGF-1 levels exhibited improved overall survival (OS) compared to those with low IGF-1 levels (p = 0.024). When stratified by regimen types, the prognostic value of high IGF-1 levels was maintained in patients treated with antiangiogenic-containing regimens (p = 0.001) but was not observed in patients treated without antiangiogenic agents (p = 0.967). Conclusions: High serum IGF-1 levels are associated with improved OS in patients with advanced HCC, but this prognostic value is confined to regimens containing antiangiogenic agents. These findings underscore the potential of IGF-1 as a biomarker for optimizing immunotherapy strategies in advanced HCC. Citation Format: Tsung-Che Wu, Ching-Tso Chen, Chien-Huai Chuang, Ann-Lii Cheng, Chih-Hung Hsu, Yu-Yun Shao. Serum Insulin-Like Growth Factor-1 levels predict outcomes in patients with advanced hepatocellular carcinoma receiving immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3318.

Abstract 3511: A T cell engager (TCE), HB134, targeting LILRB4 and CD3, exhibits potent anti-tumor activity with a favorable safety profile for monocytic AML

Abstract Background: Acute myeloid leukemia (AML), is a heterogeneous and highly aggressive malignancy, characterized by poor prognosis and limited treatment options. Monocytic AML, including M4 and M5 subtypes, presents a significant clinical challenge due to its dismal prognosis and high relapse rates post-transplant. The leukocyte immunoglobulin-like receptor B4 (LILRB4) is overexpressed in monocytic AML but is absent in normal hematopoietic stem cells, highlighting its potential as a therapeutic target. Here, we report the development of a novel TCE targeting LILRB4 and CD3, HB134, for the treatment of monocytic AML. Methods: The HB134 TCE was generated by fusing an anti-LILRB4 nanobody and an anti-CD3 Fab to a human IgG1 with a silent Fc region. Surface plasmon resonance, enzyme-linked immunosorbent assay, and flow cytometry were used to measure HB134’s binding activities to LILRB4 and CD3. Luciferase-based cellular reporter assays and a primary T cell-dependent cellular cytotoxicity (TDCC) assay using peripheral blood mononuclear cells (PBMCs) were employed to assess HB134’s T cell activation activity. Additionally, in vivo anti-tumor efficacy and pharmacokinetic (PK) profile were evaluated in mouse models. Results: HB134 exhibited sub nanomolar high binding affinity for human LILRB4 and low binding affinity to human CD3. In the cellular luciferase reporter assays, in the presence of LILRB4-positive monocytic AML cells (THP-1 or MOLM-13 cell line), HB134 induced robust T cell activation; in contrast, no T cell activation was observed in the presence of LILRB4-negative cells, indicting HB134 can specifically induce T cell activation in a LILRB4-engagement-dependent manner. In the TDCC assays using PBMCs, HB134 induced potent cytotoxicity against LILRB4-positive THP-1 and MOLM-13 cells with limited induction of cytokine release syndrome-related proinflammatory cytokines, including IL-6 and TNF-α. In an immunodeficient mouse model engrafted with human PBMCs and LILRB4-positive THP-1-luciferase cells, HB134 showed potent anti-tumor efficacy. Additionally, HB134 demonstrated a favorable PK profile in BALB/c mice. Conclusions: HB134, a TCE designed to engage T cells via CD3 and specifically target LILRB4, exhibits potent anti-tumor activity and favorable PK and safety profiles, positioning HB134 as a promising therapeutic candidate for treating LILRB4-positive monocytic AML. Citation Format: Juan Liu, Fangfang Ren, Chunmei Liu, Jing Shao, Fang Yang, Jianhe Chen, Bin Ye, Jianhua Sui. A T cell engager (TCE), HB134, targeting LILRB4 and CD3, exhibits potent anti-tumor activity with a favorable safety profile for monocytic AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3511.

Abstract 3131: In vitro analysis of collagen deposition in lung cancer-associated fibroblasts using clinically validated biomarkers

Abstract Background: Lung cancer remains the leading cause of cancer-related mortality worldwide, affecting both men and women. This high mortality rate is largely due to the frequent ineffectiveness of interventions and the late-stage diagnosis of the disease, which typically limits available treatment options. Within the tumor stroma of lung cancers, cancer-associated fibroblasts (CAFs) play a crucial role in tumor progression and metastasis by depositing extracellular matrix (ECM) components, such as collagens. To further understand lung cancer biology and support drug discovery, novel tools that replicate the tumor microenvironment are essential. In this study, we developed a pseudo-3D in vitro model incorporating healthy normal lung fibroblasts (lNFs) or lung cancer-associated fibroblasts (lCAFs), combined with clinically validated collagen-based biomarkers, as a translational platform for anti-fibrotic drug screening. Methods: Primary human CAFs from lung adenocarcinoma (lCAFs) and normal lung fibroblasts (lNFs) were cultured for 12 days in ficoll-based media supplemented with ascorbic acid under unstimulated conditions and with TGF-β stimulation. Additionally, cells were treated with the ALK5/TGF-β1 receptor kinase inhibitor (ALK5i) and Fresolimumab, a human monoclonal antibody targeting all TGF-β isoforms. Collagen type I (PRO-C1), III (PRO-C3), and VI (PRO-C6) levels were measured in the cell supernatant on days 3, 6, 9, and 12 using competitive enzyme-linked immunosorbent assay. Results: TGF-β1 stimulation increased type I, III, and VI collagen deposition in both lCAFs and lNFs. Notably, lCAFs showed higher type I collagen formation than lNFs, even after treatment. While untreated lCAFs and lNFs exhibited no type III collagen formation, TGF-β1 treatment significantly induced PRO-C3, with higher levels in lNFs than in lCAFs. PRO-C6 levels were initially comparable in untreated lCAFs and lNFs, yet TGF-β1 stimulation led to a significant increase in type VI collagen in lCAFs compared to a smaller increase in lNFs. Treatment with ALK5i and Fresolimumab reduced levels of PRO-C1, PRO-C3, and PRO-C6 in both lCAFs and lNFs. Conclusion: The pseudo-3D in vitro model using lCAFs and lNFs successfully replicates key aspects of the lung tumor microenvironment, particularly the differential collagen deposition patterns. The model demonstrated that lCAFs exhibit distinct collagen formation responses to TGF-β1, especially in type I and VI collagens, compared to lNFs. Additionally, treatment with ALK5i and Fresolimumab effectively reduced collagen formation, underscoring the model’s potential for preclinical anti-fibrotic drug screening. These findings support the model's application in studying CAF-driven ECM remodeling and assessing therapeutic responses in lung cancer. Citation Format: Annika Hettich, Morten Karsdal, Nicholas Willumsen. In vitro analysis of collagen deposition in lung cancer-associated fibroblasts using clinically validated biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3131.

Abstract 6752: Identification and validation of human antibodies targeting a novel pan-adenocarcinoma target discovered via spatial transcriptomics

Abstract Background: Adenocarcinoma (ADCA) is a type of cancer that originates in glandular epithelial cells in various organs such as pancreas, colon and lung. Spatial transcriptomics enables the high-resolution mapping of gene expression within the tumor microenvironment, preserving spatial context. This allows for the identification of tumor-specific targets in the context of complex tumor microenvironment, providing insights critical for precision oncology. Through a data-driven target discovery platform which leverages large scale transcriptomic data including ST, a novel target, POR-ADCA-001, was identified. Here, we developed human antibodies that have superior specificity and binding affinity to POR-ADCA-001. Methods: Panning phage display, filter lift assay, and Solid Phase Extraction Enzyme-Linked Immunosorbent Assay (SPE ELISA) were utilized to screen hit antibodies with strong binding potential to the target protein. Furthermore, ELISA and Surface Plasmon Resonance (SPR) analysis were used to identify lead candidate antibodies (POR-ADCA-001-01, POR-ADCA-001-02) that can effectively target POR-ADCA-001 at low concentrations. Moreover, a cell line overexpressing POR-ADCA-001 (cell-1) and a non-expressing cell line (cell-2) were validated using western blot and immunofluorescence. At last, we examined the effectiveness of the antibodies using flow cytometry, immunofluorescence, and internalization assay. Results: Spatial and differential expression analyses revealed POR-ADCA-001 as highly expressed in various ADCA tissues with low expression in normal tissues. On ELISA, 28 hits were found and the half maximal effective concentration (EC50) values of them were one or two digit nM. Among them, POR-ADCA-001-01, 02 were selected for further development and demonstrated the binding affinity of 0.84 and 14.8 nM. We demonstrated using flow cytometry and immunofluorescence that the two antibodies bind strongly to cell-1 but hardly bind to cell-2. Internalization assays confirmed substantial uptake of the antibodies in cell-1, with minimal activity in control cell lines. Conclusion: Our research identified POR-ADCA-001 as a target specific to pan-adenocarcinoma and discovered two human IgG antibodies that demonstrate high specificity and effective internalization in cells that express the target. These antibodies could be utilized for the development of targeted therapeutics such as antibody-drug conjugates (ADCs) or radiopharmaceutical therapies (RPTs). This study emphasizes the promise of ST in uncovering new therapeutic targets and improving cancer treatment. Citation Format: MinKyu Kim, Jinyeoung Choi, Jae Eun Lee, Hongyoon Choi, Hyung-Jun Im. Identification and validation of human antibodies targeting a novel pan-adenocarcinoma target discovered via spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6752.

Abstract 140: Profiling ECM biomarkers PRO-C3 and PRO-C5 in plasma from patients with esophagogastric cancer

Abstract Background: Esophagogastric cancer encompasses malignancies of the esophagus, esophagogastric junction, and stomach. These cancers involve distinct regions of the esophagus and gastroesophageal junction (GEJ) and exhibit unique clinicopathologic, histologic, and mutational features. The major constituents of the tumor microenvironment (TME) are besides the tumor cells themselves, cancer associated fibroblasts (CAFs), stromal cells and the extracellular matrix (ECM). CAFs synthesize excess amounts of collagens leading to a fibrotic TME with poor response to anti-cancer treatment; it was previously found that among other types of collagens COL3A1 and COL5A1 are expressed in most of activated CAFs. The aim of this study was to investigate the relevance of non-invasive serological biomarkers of type III collagen synthesis (PRO-C3) and type V collagen synthesis (PRO-C5) in patients with GEAC and ESCC. Methods: Levels of PRO-C3 and PRO-C5 were measured non-invasively using competitive enzyme-linked immunosorbent assays in EDTA Plasma from patients diagnosed with esophageal squamous cell carcinoma (ESCC) (n=27), gastroesophageal adenocarcinoma (GEAC) (n=165) and compared to healthy controls (n=20). Blood samples were obtained during biopsy, and the biomarker measurements reflect baseline levels. PRO-C3 and PRO-C5 levels in patients with GEAC and ESCC were assessed according to disease stage, treatment type and resection status. Results: Circulating PRO-C3 levels were significantly elevated in both GEAC (p = 0.0025) and ESCC (p = 0.0022), while PRO-C5 levels were markedly increased in both subtypes (GEAC: p = 0.0012; ESCC: p = 0.0002). PRO-C5 levels were higher in ESCC compared to GEAC (p = 0.0023), whereas PRO-C3 levels were similar between the two (p = 0.441). Stratifying by treatment intention showed no significant differences in PRO-C3 or PRO-C5 levels in ESCC patients receiving curative or palliative care. In GEAC, PRO-C5 levels were higher in palliative treatment patients (p = 0.01). For resection status, neither PRO-C3 nor PRO-C5 levels varied significantly in GEAC. In ESCC, PRO-C3 levels were elevated in non-resected samples (p = 0.002). Stratification by cancer stage revealed significant increases in PRO-C5 levels in GEAC patients at Stage III (p = 0.002) and Stage IV (p = 0.004). Conclusion: The non-invasive fibrosis-associated biomarkers PRO-C3 and PRO-C5, indicative of type III and type V collagen synthesis mediated by activated CAFs, are elevated in patients with ESCC and GEAC. These biomarkers show promise as non-invasive tools for stratifying and monitoring patients across different subtypes of esophagogastric cancer. Citation Format: Eleonora Moroncini, Andrea Ponzetta, Magnus Nilsson, Fredrik Klevebro, Rebecca Maltez de Sousa, Nora Norén, Morten Karsdal, Nicholas Willumsen. Profiling ECM biomarkers PRO-C3 and PRO-C5 in plasma from patients with esophagogastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 140.

The role of serum and seminal fluid anti-müllerian hormone (AMH) in differentiation subtypes of male infertility

Background Infertility is the inability to conceive after one year of unprotected intercourse, with male factors accounting for about 50% of cases. Anti-Müllerian hormone, a glycoprotein belongs to Transforming growth factor beta, is secreted from Sertoli cells and plays a key role in male sexual differentiation. It serves as a biomarker reflect Sertoli-cell function and spermatogenic capacity. Objectives The study was designed for examine the potential role of anti-müllerian hormone in differentiating the subtypes of male-infertility. Methods This case-control study was done at the infertility center of Al-Batool Teaching Hospital in Diyala Governorate, Iraq, by the Department of Biochemistry / College of Medicine / University of Baghdad between April 2024 and January 2025. It included 111 males, aged (20-55 years). These subjects were subdivided into four groups based on seminal fluid analysis: a normozoospermic group, which included 30 males and served as a control group. asthenozoospermia group, which involved 29 males; Group 3 comprised 25 patients with oligozoospermia; and Group 4 involved 27 subjects with idiopathic (unexplained) infertility. Investigations included serum and seminal fluid measurements of anti-müllerian hormone using enzyme-linked immunosorbent assays. Results The results showed that the mean, (±SD) values of anti-müllerian hormone concentrations were significantly increased in the serum compared with the results of seminal plasma in all groups except the finding in the oligozoospermia group were anti-müllerian hormone higher than in serum. The reservoir operating characteristics (ROC) analysis revealed that Serum AMH exhibited fair to good diagnostic performance, while seminal fluid AMH displayed lower diagnostic accuracy in distinguishing subtypes of male infertility. Conclusion The outcomes suggest that serum AMH may serve as a valuable biomarker in the differentiation of male-infertility subtypes. Keywords: Anti-müllerian hormone, Asthenozoospermia, Oligozoospermia, Plasma Seminal fluid, Unexplained infertility.

Investigation of PANoptosis pathway in age-related macular degeneration triggered by Aβ1-40

Our study aimed to identify PANoptosis in Aβ1-40-induced AMD, both in vivo and in vitro, and to determine if AIM2-PANoptosome mediates this process. We used transcriptomics to explore the signaling pathways and target genes linked to PANoptosis within a mouse model of AMD triggered by Aβ1-40. Optical coherence tomography (OCT), hematoxylin and eosin (H&E) staining, and electroretinography (ERG) were employed to assess retinal damage in terms of morphology and function. Morphological changes in ARPE-19 cells were observed using optical microscopy and scanning electron microscopy. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of cytokines in cell supernatants, mouse orbital serum, and human plasma to evaluate the severity of inflammation. CO-immunoprecipitation(CoIP) and molecular docking were performed to assess the impact and expression of proteins associated with the AIM2-PANoptosome. Quantitative polymerase chain reaction (qPCR), Western blot (WB), immunofluorescence, and apoptosis detection kits were used to evaluate the expression levels of genes and proteins related to PANoptosis-like cell death. Our results showed that the Aβ1-40-induced AMD model had increased expression of apoptosis, necroptosis, and pyroptosis pathways, and AIM2-PANoptosome components. CoIP and docking confirmed increased AIM2, ZBP1, and PYRIN levels under Aβ1-40 treatment. WB and immunofluorescence showed upregulation of PANoptosis-related proteins. Inhibitors reduced Aβ-induced protein expression. ELISA showed increased inflammatory cytokines. Apoptosis assays and microscopy revealed Aβ1-40-induced ARPE-19 cell loss and morphological changes. In conclusion, the Aβ1-40-induced AMD model displayed PANoptosis-like cell death, offering insights into disease pathogenesis.

Anti-mullerian hormone in felids: A systematic review.

Anti-mullerian hormone in felids: A systematic review.

OGA promotes human dental pulp stem cell senescence and inhibits mitophagy by inhibition of O-GlcNAcylation of KLF2

BackgroundDental pulp stem cells (DPSCs) aging impedes its application in tooth regeneration techniques, involving abnormal mitophagy. O-GlcNAcylation is a post-translational modification that regulates various cellular processes. Here, we aimed to investigate the role of O-GlcNAcylation in mitophagy and senescence.MethodsDPSCs were cultured and passaged in vitro, and the 7th (p7) and 15th (p15) generation cells were collected. OGA and KLF2 were knocked down in p15 cells. Cell senescence was evaluated using senescence associated β-galactosidase staining, enzyme-linked immunosorbent assay, and western blotting; mitophagy was evaluated using western blotting. The regulation of OGA on the O-GlcNAcylation of KLF2 was analyzed using immunoprecipitation and western blotting.ResultsThe results showed that p15 cells were more senescent than p7 cells and had poor mitophagy, with the higher expression of OGA. Knockdown of OGA inhibited senescence and promoted mitophagy in DPSCs. Moreover, silencing of KLF2 reversed the effects on senescence and mitophagy mediated by OGA knockdown. Additionally, OGA suppressed the O-GlcNAcylation of KLF2 at S177 site and thus reduced its stability.ConclusionSilencing of OGA promotes mitophagy and inhibits DPSC senescence by promoting the O-GlcNAcylation of KLF2, suggesting a novel mechanism underlying DPSC senescence.

Increased level of serum asymmetric dimethylarginine in individuals with more severe cognitive impairment, as evaluated using Montreal Cognitive Assessment instead of Mini-Mental State Examination

BackgroundThis study aimed to explore the link between cognitive impairment and levels of asymmetric dimethylarginine (ADMA).MethodsThe study included 172 patients from the Department of Geriatrics and Neurology at the Second Affiliated Hospital of Harbin Medical University. The enrollment period spanned from October 2013 to July 2014. To assess their cognitive function, we used the Mini-Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA). Additionally, automatic biochemical analyzers were employed to measure various biochemical blood indexes, while enzyme-linked immunosorbent assay was used to determine the serum ADMA concentrations.ResultsThe participants were categorized into four groups based on the MMSE scale, which reflects cognition (higher scores indicating better cognitive function), and five groups based on the MoCA scale, which also measures cognition (higher scores indicating better cognitive function). Various factors were analyzed for their statistical significance in relation to different cognitive impairment groups determined by each scale. Regarding the MoCA scale, the following factors were found to be statistically significant: Age (P = 0.0001), systolic blood pressure (P = 0.0261), ALT (P = 0.0104), AST (P = 0.0106), endogenous creatinine clearance (P = 0.0006), and serum ADMA concentration (P = 0.0383). For the MMSE scale, the following factors showed statistical significance: Age (P = 0.0008), ALT (P = 0.0002), AST (P = 0.0088), CRP (P = 0.0407), and endogenous creatinine clearance (P = 0.0027). Interestingly, as the scores on the MoCA scale decreased, the serum ADMA concentration increased (P=0.0383), but this trend was not observed in the groups classified based on the MMSE scale (P > 0.05).ConclusionThe level of sensitivity measured by the MoCA scale indicated the presence of initial cognitive dysfunction. The extent of cognitive impairment showed a direct correlation with ADMA levels, indirectly implying a connection between impaired endothelial function and cognitive dysfunction.