Antibody Array Research Articles

STEM-01. DEVELOPMENT OF A HUMAN-SPECIFIC 3DIN VITRO PAEDIATRIC CEREBELLAR TUMOUR MODEL USING DECELLULARISED BRAIN AUTOPSY TISSUE

Abstract BACKGROUND Medulloblastoma (MB) and Atypical Teratoid/ Rhabdoid Tumours (ATRT) represent paediatric cerebellar tumours with a dismal prognosis. For ATRT in particular, 5-year survival remains at less than 32%. We have developed a 3D in vitro model termed ‘tumoursphere matrix’ which recapitulates in vivo crosstalk between tumour and reactive brain. METHODS AND RESULTS We co-cultured D283 (Group 3-MYC amplified MB) and BT12 (ATRT-MYC) cells with primary human cerebellar astrocytes within a PEGDA hydrogel containing decellularised cerebellar extracellular matrix (ECM) derived from non-disease human autopsy brain. Cerebellar ECM was extensively characterised, showing a significant reduction in cellular material whilst retaining ECM ligands. Reduction of DNA content to 174 ng/mg was corroborated by an absence of nuclei in immunohistochemical staining. Colorimetric assays indicated collagen and glycosaminoglycan retention in decellularised ECM, and Immunofluorescence confirmed retention of fibronectin, laminin and hyaluronic acid. Detection of all ECM ligands was cross-validated by Orbitrap-Secondary Ion Mass Spectrometry. A solubilised ECM/PEGDA hydrogel was used to coat an AggreWell™ plate. Primary human cerebellar astrocytes, D283 and BT12 cells were cultured as spheroids within the ECM/PEGDA hydrogel for 7 days with no significant decrease in cellular viability. Cytokine and Human Matrix Metalloproteinase (MMP) Antibody Arrays showed cytokine and MMP expression in all three cell lines was retained in the presence of ECM material. Furthermore, D283 and BT12 cells were co-cultured with primary astrocytes for 7 days prior to separation using fluorescence activated cell sorting to generate individual cell populations. CONCLUSIONS We have developed a 3D in vitro model which can co-culture cells for over 7 days, whilst recapitulating the in vivo tumour environment. RNA sequencing is currently in progress to identify differentially expressed genes in cerebellar tumour cells upon astrocytic and brain ECM crosstalk. We hypothesise that biochemical signalling underlying this communication will reveal putative therapeutic vulnerabilities.

Neurotrophin-3 Facilitates Stemness Properties and Associates with Poor Survival in Lung Cancer

Introduction: Cancer stem cells (CSCs) shape the tumor microenvironment via neuroendocrine signaling and orchestrate drug resistance and metastasis. Cytokine antibody array demonstrated the upregulation of neurotrophin-3 (NT-3) in lung CSCs. This study aims to dissect the role of NT-3 in lung CSCs during tumor innervation. Methods: Western blotting, quantitative reverse transcription-PCR, and flow cytometry were used to determine the expression of the NT-3 axis in lung CSCs. NT-3-knockdown and NT-3-overexpressed cells were derived lung CSCs, followed by examining the stemness gene expression, tumorsphere formation, transwell migration and invasion, drug resistance, soft agar colony formation, and in vivo tumorigenicity. Human lung cancer tissue microarray and bioinformatic databases were used to investigate the clinical relevance of NT-3 in lung cancer. Results: NT-3 and its receptor tropomyosin receptor kinase C (TrkC) were augmented in lung tumorspheres. NT-3 silencing (shNT-3) suppressed the migration and anchorage-independent growth of lung cancer cells. Further, shNT-3 abolished the sphere-forming capability, chemo-drug resistance, invasion, and in vivo tumorigenicity of lung tumorspheres with a decreased expression of CSC markers. Conversely, NT-3 overexpression promoted migration and anchorage-independent growth and fueled tumorsphere formation by upregulating the expression of CSC markers. Lung cancer tissue microarray analysis revealed that NT-3 increased in patients with advanced-stage, lymphatic metastasis and positively correlated with Sox2 expression. Bioinformatic databases confirmed a co-expression of NT-3/TrkC-axis and demonstrated that NT-3, NT-3/TrkC, NT-3/Sox2, and NT-3/CD133 worsen the survival of lung cancer patients. Conclusion: NT-3 conferred the stemness features in lung cancer during tumor innervation, which suggests that NT-3-targeting is feasible in eradicating lung CSCs.

Differential Inflammatory Cytokine Elaboration in Serum from Brick Kiln Workers in Bhaktapur, Nepal.

Previous studies involving workers at brick kilns in the Kathmandu Valley of Nepal have investigated chronic exposure to hazardous levels of fine particulate matter (PM2.5) common in ambient and occupational environments. Such exposures are known to cause and/or exacerbate chronic respiratory diseases, including chronic obstructive pulmonary disease (COPD) and asthma. However, there is a paucity of data regarding the status of systemic inflammation observed in exposed workers at brick manufacturing facilities within the country. In the current study, we sought to elucidate systemic inflammatory responses by quantifying the molecular cytokine/chemokine profiles in serum from the study participants. A sample of participants were screened from a kiln in Bhaktapur, Nepal (n = 32; 53% female; mean ± standard deviation: 28.42 ± 11.47 years old) and grouped according to job category. Blood was procured from participants on-site, allowed to clot at room temperature, and centrifuged to obtain total serum. A human cytokine antibody array was used to screen the inflammatory mediators in serum samples from each of the participants. For the current study, four job categories were evaluated with n = 8 for each. Comparisons were generated between a control group of administration workers vs. fire master workers, administration workers vs. green brick hand molders, and administration workers vs. top loaders. We discovered significantly increased concentrations of eotaxin-1, eotaxin-2, GCSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-6, IL-8, TGF-β1, TNF-α, and TIMP-2 in serum samples from fire master workers vs. administration workers (p < 0.05). Each of these molecules was also significantly elevated in serum from green brick hand molders compared to administration workers (p < 0.05). Further, each molecule in the inflammatory screening with the exception of TIMP-2 was significantly elevated in serum from top loaders compared to administration workers (p < 0.05). With few exceptions, the fire master workers expressed significantly more systemic inflammatory molecular abundance when compared to all other job categories. These results reveal an association between pulmonary exposure to PM2.5 and systemic inflammatory responses likely mediated by cytokine/chemokine elaboration. The additional characterization of a broader array of inflammatory molecules may provide valuable insight into the susceptibility to lung diseases among this population.

Zedoary turmeric oil injection ameliorates lung inflammation via platelet factor 4 and regulates gut microbiota disorder in respiratory syncytial virus-infected young mice

BackgroundRespiratory syncytial virus (RSV)-induced lung inflammation is one of the main causes of hospitalization and easily causes disruption of intestinal homeostasis in infants, thereby resulting in a negative impact on their development. However, the current clinical drugs are not satisfactory. Zedoary turmeric oil injection (ZTOI), a patented traditional Chinese medicine (TCM), has been used for clinical management of inflammatory diseases. However, its in vivo efficacy against RSV-induced lung inflammation and the underlying mechanism remain unclear.PurposeThe present study was designed to confirm the in vivo efficacy of ZTOI against lung inflammation and intestinal disorders in RSV-infected young mice and to explore the potential mechanism.Study design and methodsLung inflammation was induced by RSV, and cytokine antibody arrays were used to clarify the effectiveness of ZTOI in RSV pneumonia. Subsequently, key therapeutic targets of ZTOI against RSV pneumonia were identified through multi-factor detection and further confirmed. The potential therapeutic material basis of ZTOI in target tissues was determined by non-target mass spectrometry. After confirming that the pharmacological substances of ZTOI can reach the intestine, we used 16S rRNA-sequencing technology to study the effect of ZTOI on the intestinal bacteria.ResultsIn the RSV-induced mouse lung inflammation model, ZTOI significantly reduced the levels of serum myeloperoxidase, serum amyloid A, C-reactive protein, and thymic stromal lymphoprotein; inhibited the mRNA expression of IL-10 and IL-6; and decreased pathological changes in the lungs. Immunofluorescence and qPCR experiments showed that ZTOI reduced RSV load in the lungs. According to cytokine antibody arrays, platelet factor 4 (PF4), a weak chemotactic factor mainly synthesized by megakaryocytes, showed a concentration-dependent change in lung tissues affected by ZTOI, which could be the key target for ZTOI to exert anti-inflammatory effects. Additionally, sesquiterpenes were enriched in the lungs and intestines, thereby exerting anti-inflammatory and regulatory effects on gut microbiota.ConclusionZTOI can protect from lung inflammation via PF4 and regulate gut microbiota disorder in RSV-infected young mice by sesquiterpenes, which provides reference for its clinical application in RSV-induced lung diseases.

Effects of radiation mitigating amino acid mixture on mice of different sexes.

To date, few FDA-approved medical countermeasures are available for addressing hematopoietic acute radiation syndrome (H-ARS). In this study, we present our latest research findings focusing on the evaluation of a novel radiation mitigator known as the mitigating amino acid mixture (MAAM). MAAM is composed of five amino acids as the recently reported amino acid-based oral rehydration solution for mitigating gastrointestinal (GI)-ARS. CD2F1 male and female mice were exposed to 60Co-γ total body irradiation (TBI) at 9.0 or 9.5 Gy. Following irradiation, mice were orally administered with MAAM or a saline vehicle control once daily for a duration of 14 days, commencing 24 h after TBI. Mouse survival and body weight change were monitored for 30 days after irradiation. Complete blood counts (CBCs), bone marrow (BM) stem and progenitor cell survival (clonogenicity), and a serum cytokine antibody array were analyzed using samples from day 30 surviving mice. Our data revealed that MAAM treatment significantly enhanced survival rates in irradiated male CD2F1 mice, and the survival rate increased from 25% in the vehicle control group to 60% in the MAAM-treated group (p < 0.05) after 9.0 Gy TBI. The number of BM colonies significantly increased from 41.8 ± 6.4 /104 cells (in the vehicle group) to 78.5 ± 17.0 /104 cells (in the MAAM group) following 9.0 Gy TBI. Furthermore, MAAM treatment led to a decrease in the levels of six cytokines/proteins [cluster of differentiation 40 (CD40), interleukin (IL)-17A, C-X-C motif chemokine 10 (CXCL10/CRG-2), cutaneous T cell-attracting chemokine (CTACK), macrophage inflammatory protein (MIP)-3β, and IL-1β] and an increase in the levels of five other cytokines/proteins [IL-3Rβ, IL-5, leptin, IL-6, and stem cell factor (SCF)] in mouse serum compared to the vehicle group after 9.0 Gy TBI. However, similar alleviating effects of MAAM were not observed in the irradiated CD2F1 female mice. The serum cytokine profile in the irradiated female mice was different compared to the irradiated male mice. In summary, our data suggest that the beneficial effects of the mitigative amino acid combination treatment after radiation exposure may depend on sex.

Angiogenic protein profiling, phytochemical screening and in silico anti-cancer targets validation of stem, leaves, fruit, and seeds of Calotropis procera in human liver and breast cancer cell lines

The main focus of anticancer drug discovery is on developing medications that are gentle on normal cells and should have the ability to target multiple anti-cancer pathways. Liver cancer is becoming a worldwide epidemic due to the highest occurring and reoccurring rate in some countries. Calotropis procera is a xerophytic herbal plant growing wildly in Saudi Arabia. Due to its anti-angiogenic and anticancer capabilities, “C. procera” is a viable option for developing innovative anticancer medicines. However, no study has been done previously, to discover angiogenic and anti-cancer targets which are regulated by C. procera in liver cancer. In this study, leaves, stems, flowers, and seeds of C. procera were used to prepare crude extracts and were fractionated into four solvents of diverse polarities. These bioactivity-guided solvent fractions helped to identify useful compounds with minimal side effects. The phytoconstituents present in the leaves and stem were identified by GC-MS. In silico studies were done to predict the anti-cancer targets by major bioactive constituents present in leaves and stem extracts. A human angiogenesis antibody array was performed to profile novel angiogenic targets. The results from this study showed that C. procera extracts are an ideal anti-cancer remedy with minimum toxicity to normal cells as revealed by zebrafish in vivo toxicity screening assays. The novel antiangiogenic and anticancer targets identified in this study could be explored to design medication against liver cancer.

Anticancer Activity and Molecular Mechanisms of Acetylated and Methylated Quercetin in Human Breast Cancer Cells

Quercetin, a flavonoid polyphenol found in many plants, has garnered significant attention due to its potential cancer chemoprevention. Our previous studies have shown that acetyl modification of the hydroxyl group of quercetin altered its antitumor effects in HepG2 cells. However, the antitumor effect in other cancer cells with different gene mutants remains unknown. In this study, we investigated the antitumor effect of quercetin and its methylated derivative 3,3′,4′,7-O-tetramethylquercetin (4Me-Q) and acetylated derivative 3,3′,4′,7-O-tetraacetylquercetin (4Ac-Q) on two human breast cancer cells, MCF-7 (wt-p53, caspase-3-ve) and MDA-MB-231 (mt-p53, caspase-3+ve). The results demonstrated that 4Ac-Q exhibited significant cell proliferation inhibition and apoptosis induction in both MCF-7 and MDA-MB-231 cells. Conversely, methylation of quercetin was found to lose the activity. The human apoptosis antibody array revealed that 4Ac-Q might induce apoptosis in MCF-7 cells via a p53-dependent pathway, while in MDA-MB-231 cells, it was induced via a caspase-3-dependent pathway. Furthermore, an evaluation using a superoxide inhibitor, MnTBAP, revealed 4Ac-Q-induced apoptosis in MCF-7 cells in a superoxide-independent manner. These findings provide valuable insights into the potential of acetylated quercetin as a new approach in cancer chemoprevention and offer new avenues for health product development.

The serine protease CORIN promotes progression of gastric cancer by mediating the ERK1/2 MAPK pathway.

The serine protease CORIN catalyzes pro-atrial natriuretic peptide (pro-ANP) into an active ANP and maintains homeostasis of the internal environment. However, it is unclear whether CORIN participates in the regulation of tumor progression. We analyzed the expression profile of CORIN in gastric cancer tissues (GCs) and adjacent nontumoral tissues (NTs). We investigated the prognostic value of CORIN in GC patients. We characterized the in vitro and in vivo activity of CORIN in cultured GC cells with gain-of-function and loss-of-function experiments. The underlying mechanism was explored by using bioinformatics, a signaling antibody array, and confirmative western blot analyses, as well as rescue experiments with highly selective small-molecule inhibitors targeting the ERK1/2 MAPK signaling pathway. CORIN was upregulated in GCs than in NTs. Overexpression of CORIN was correlated with unfavorable prognoses in patients with GC. Ectopic expression of CORIN was promoted, whereas silencing of CORIN suppressed proliferation, colony formation, migration and invasion of GC cells, and tumor growth in vivo. Overexpression of CORIN-induced epithelial-mesenchymal transition (EMT) and activation of the ERK1/2 MAPK signaling pathway, while silencing of CORIN yielded opposite results. The in vitro tumor-promoting potency of CORIN could be antagonized by selective inhibitors targeting the ERK1/2 MAPK pathway. In conclusion, CORIN is a potential prognostic marker and therapeutic target for GC patients, which may promote tumor progression by mediating the ERK1/2 MAPK signaling pathway and EMT in GC cells.

Combinatory Nanovesicle with siRNA-Loaded Extracellular Vesicle and IGF-1 for Osteoarthritis Treatments.

Extracellular vesicles (EVs) have been found to have the characteristics of their parent cells. Based on the characteristics of these EVs, various studies on disease treatment using mesenchymal stem cell (MSC)-derived EVs with regenerative activity have been actively conducted. The therapeutic nature of MSC-derived EVs has been shown in several studies, but in recent years, there have been many efforts to functionalize EVs to give them more potent therapeutic effects. Strategies for functionalizing EVs include endogenous and exogenous methods. In this study, human umbilical cord MSC (UCMSC)-derived EVs were selected for optimum OA treatments with expectation via bioinformatics analysis based on antibody array. And we created a novel nanovesicle system called the IGF-si-EV, which has the properties of both cartilage regeneration and long-term retention in the lesion site, attaching positively charged insulin-like growth factor-1 (IGF-1) to the surface of the UCMSC-derived Evs carrying siRNA, which inhibits MMP13. The downregulation of inflammation-related cytokine (MMP13, NF-kB, and IL-6) and the upregulation of cartilage-regeneration-related factors (Col2, Acan) were achieved with IGF-si-EV. Moreover, the ability of IGF-si-EV to remain in the lesion site for a long time has been proven through an ex vivo system. Collectively, the final constructed IGF-si-EV can be proposed as an effective OA treatment through its successful MMP13 inhibition, chondroprotective effect, and cartilage adhesion ability. We also believe that this EV-based nanoparticle-manufacturing technology can be applied as a platform technology for various diseases.

Polyphyllin I ameliorates gefitinib resistance and inhibits the VEGF/VEGFR2/p38 pathway by targeting HIF-1a in lung adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) is the most common pathological type of lung cancer. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been administered as the first-line therapy for patients with EGFR mutations in LUAD, but it is almost inevitable that resistance to EGFR-TKIs therapy eventually arises. Polyphyllin I (PPI), derived from Paris polyphylla rhizomes, has been shown to have potent anti-cancer properties in a range of human cancer types including LUAD. However, the role of PPI in gefitinib resistance and the underlying mechanism remain elusive. PurposeTo evaluate the antitumor impacts of PPI on gefitinib resistance cells and investigate its molecular mechanism. MethodsCCK-8, wound healing, transwell assay, and xenograft model were performed to determine the anti-cancer effects of PPI as well as its ability to overcome gefitinib resistance. Immunoblotting, co-immunoprecipitation, phospho-RTK antibody array, qRT-PCR, and immunofluorescence were utilized to explore the mechanism by which PPI overrides gefitinib resistance. ResultsPPI inhibited cell survival, growth, and migration/invasion in both gefitinib-sensitive (PC9) and -resistant (PC9/GR) LUAD cells (IC50 at 2.0 μM). Significantly, treatment with PPI at 1.0 μM resensitized the resistant cells to gefitinib. Moreover, cell-derived xenograft experiments revealed that the combination of PPI and gefitinib overcame gefitinib resistance. The phospho-RTK array and immunoblotting analyses showed PPI significant inhibition of the VEGFR2/p38 pathway. In addition, molecular docking suggested the interaction between PPI and HIF-1α. Mechanistically, PPI reduced the protein expression of HIF-1α in both normoxia and hypoxia conditions by triggering HIF-1α degradation. Moreover, HIF-1α protein but not mRNA level was elevated in gefitinib-resistant LUAD. We further demonstrated that PPI considerably facilitated the binding of HIF-1α to VHL. ConclusionsWe present a novel discovery demonstrating that PPI effectively counteracts gefitinib resistance in LUAD by modulating the VEGF/VEGFR2/p38 pathway. Mechanistic investigations unveil that PPI facilitates the formation of the HIF-1α /VHL complex, leading to the degradation of HIF-1α and subsequent inhibition of angiogenesis. These findings uncover a previously unidentified mechanism governing HIF-1α expression in reaction to PPI, providing a promising method for therapeutic interventions targeting EGFR-TKI resistance in LUAD.

Differential serum inflammatory cytokine elaboration in Nepali brick factory workers

Previous studies involving workers at brick kilns in the Kathmandu Valley of Nepal have revealed chronic exposure to hazardous levels of fine particulate matter (PM2.5) common in ambient and occupational environments. Such exposures are known to cause and/or exacerbate chronic respiratory diseases including chronic obstructive pulmonary disease (COPD). However, there is a paucity of data regarding the systemic inflammatory status of exposed workers at brick manufacturing facilities. In the current study, we sought to elucidate systemic inflammatory responses by quantifying the molecular cytokine/chemokine profiles in serum from study participants. A sample of participants (N = 48; 54% female; 31.77±9.76 years-old) were distributed among four different job categories. Blood was procured from participants on-site, allowed to clot at room temperature, and centrifuged to obtain total serum. A human cytokine antibody array was used to screen cytokines/chemokines in serum samples. Comparisons were generated between a control group of administration workers vs. fire master workers, administration vs. green brick hand molders, and administration vs. top loaders. We discovered significantly increased concentrations of eotaxin-1, eotaxin-2, GCSF, GM-CSF, IFN-γ, IL-1γα, IL-1β, IL-6, IL-8, TGF-β1, TNF-α, and TIMP-2 in serum samples from fire master workers vs. administration (p &lt; 0.03). Each of these molecules, with the exception of TIMP-2, were also significantly elevated in the serum from green brick hand molders (p &lt; 0.02). In addition, each molecule was significantly elevated in serum from top loaders compared to administration workers (p &lt; 0.03). With few exceptions, the fire master workers expressed significantly more systemic inflammatory molecular abundance when compared to all other job descriptions. These results reveal an association between pulmonary exposure to PM2.5 and systemic inflammatory responses likely mediated by cytokine/chemokine elaboration. Additional characterization of a broader array of inflammatory molecules may provide valuable insight into susceptibility to lung diseases including COPD. This work was supported in part by funding from Undergraduate Mentoring and a Widtsoe Research Grant from Brigham Young University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Interleukin-15 and Interleukin-8 Axis as a Novel Mechanism for Recurrent Chronic Rhinosinusitis with Nasal Polyps.

The prevention of postoperative recurrence after endoscopic sinus surgery (ESS) relies on targeting specific pathological mechanisms according to individuals' immunological profiles. However, essential biomarkers and biological characteristics of difficult-to-treat chronic rhinosinusitis (CRS) patients are not well-defined. The aim of this study was to explore the immunologic profiles of subgroups of CRS patients and determine the specific cytokines responsible for recalcitrant or recurrent CRS with nasal polyposis (rCRSwNP). We used 30 cytokine antibody arrays to determine the key cytokines related to recurrent polypogenesis. Enzyme-linked immunosorbent assay (ELISA) experiments were conducted to assess the levels of these key cytokines in 78 patients. Polymorphonuclear leukocytes (PMNs) isolated from nasal polyps were challenged with specific cytokines to examine the levels of enhanced interleukin (IL)-8 production. Finally, we used immunohistochemistry (IHC) staining to check for the presence and distribution of the biomarkers within nasal polyps. A cytokine antibody array revealed that IL-8, IL-13, IL-15, and IL-20 were significantly higher in the recalcitrant CRSwNP group. Subsequent ELISA screening showed a stepwise increase in tissue IL-8 levels in the CHR, CRSsNP, and CRSwNP groups. PMNs isolated from nine CRSwNP cases all demonstrated enhanced IL-8 production after IL-15 treatment. IHC staining was labeled concurrent IL-8 and IL-15 expression in areas of prominent neutrophil infiltration. Our results suggest that IL-15 within the sinonasal mucosa plays a crucial role in promoting IL-8 secretion by infiltrating PMNs in recalcitrant nasal polyps. In addition, we propose a novel therapeutic strategy targeting the anti-IL-15/IL-8 axis to treat CRS with nasal polyposis.

Phosphatidylinositol 4-Kinase III Alpha Governs Cytoskeletal Organization for Invasiveness of Liver Cancer Cells

High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition, hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to hepatocellular carcinoma progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process. Several hepatic cell culture and mouse models were used to study the functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing, and PI4KIIIα-specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. High PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased phosphorylation of paxillin and cofilin. This led to morphologic alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver-specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane phosphatidylinositol 3,4-bisphosphate-enriched, invadopodia-like structures that regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. PI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to the progression of liver cancer and identify promising targets for therapeutic intervention.

Ketohexokinase-A deficiency attenuates the proliferation via reducing β-catenin in gastric cancer cells

Overconsumption of fructose is closely related to cancer. Ketohexokinase (KHK) catalyzes the conversion from fructose to fructose-1-phosphate (F1P), which is the first and committed step of fructose metabolism. Recently, aberrant KHK activation has been identified in multiple malignancies. However, the roles of KHK in gastric cancer (GC) cells are largely unclear. Herein, we reveal that the expression of ketohexokinase-A (KHK-A), one alternatively spliced KHK isoform that possesses low affinity for fructose, was markedly increased in GC cells. Depletion of endogenous KHK-A expression using lentiviruses encoding short hairpin RNAs (shRNAs) or pharmaceutical disruption of KHK-A activity using KHK-IN-1 hydrochloride in GC NCI–N87 and HGC-27 cells inhibited the proliferation in vitro and in vivo. Additionally, the mitochondrial respiration in the GC cells with KHK-A deficiency compared with the control cells was significantly impaired. One commercially-available antibody array was used to explore the effects of KHK-A knockdown on signaling pathways, showing that β-catenin was remarkably reduced in the KHK-A deficient GC cells compared with the control ones. Pharmaceutical reduction in β-catenin levels slowed down the proliferation of GC cells. These data uncover that KHK-A promotes the proliferation in GC cells, indicating that this enzyme might be a promising therapeutical target for GC treatment.

Farnesol and Selected Nanoparticles (Silver, Gold, Copper, and Zinc Oxide) as Effective Agents Against Biofilms Formed by Pathogenic Microorganisms.

Biofilms, which are created by most microorganisms, are known for their widely developed drug resistance, even more than planktonic forms of microorganisms. The aim of the study was to assess the effectiveness of agents composed of farnesol and nanoparticles (silver, gold, copper, and zinc oxide) in the degradation of biofilms produced by pathogenic microorganisms. Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans were used to create the biofilm structure. Colloidal suspensions of silver, gold, copper, and zinc oxide (Ag, Au, Cu, ZnO) with the addition of farnesol (F) were used as the treatment factor. The size distribution of those composites was analyzed, their zeta potential was measured, and their structure was visualized by transmission electron microscopy. The viability of the microorganism strains was assessed by an XTT assay, the ability to form biofilms was analyzed by confocal microscopy, and the changes in biofilm structure were evaluated by scanning electron microscopy. The general toxicity toward the HFFF2 cell line was determined by a neutral red assay and a human inflammation antibody array. The link between the two components (farnesol and nanoparticles) caused mutual stability of both components. Planktonic forms of the microorganisms were the most sensitive when exposed to AgF and CuF; however, the biofilm structure of all microorganism strains was the most disrupted (both inhibition of formation and changes within the structure) after AgF treatment. Composites were not toxic toward the HFFF2 cell line, although the expression of several cytokines was higher than in the not-treated group. The in vitro studies demonstrated antibiofilm properties of composites based on farnesol and nanoparticles. The greatest changes in biofilm structure were triggered by AgF, causing an alteration in the biofilm formation process as well as in the biofilm structure.

Abstract 3178: Vitamin B1 derivative, fursultiamine, prevents colon cancer growth

Abstract Colon cancer is the second deadliest type of cancer in the United States, with surgical resection being the primary treatment modality for localized early-stage colon cancer. Still, more treatment and prevention options are needed to help improve the outcomes for colon cancer as they are currently limited to invasive procedures. One promising option is fursultiamine, a disulfide lipid-soluble vitamin B1 derivative, which has anti-oxidative and anti-inflammatory activity that could aid in preventing colon cancer. We hypothesize that supplementation of fursultiamine could be an effective chemopreventive treatment to control the growth and spread of colon carcinogenesis. Human colorectal adenocarcinoma (SW480) cell lines were incubated with complete media (CM) or human recombinant endothelial growth factor (EGF 20 ng/ml) in the absence and presence of fursultiamine. The cell viability was examined by MTT assay in a concentration- (0-150 uM) and time (24 and 48 h) - dependent manner. Our results indicate that fursultiamine prevents SW480 cell death in a dose-dependent manner. Apoptosis was detected by using Annexin-V staining and live and death cell assay. Our results suggest that fursultiamine prevents SW480 cell proliferation by inducing apoptotic cell death. The production of reactive oxygen species (ROS) and activation of caspase-3 were examined by specific assay kits. Treatment with fursultiamine resulted in the decreased production of ROS and activation of caspase-3. The expression of anti-apoptotic, pro-apoptotic, and pro-inflammatory factors was analyzed using antibody arrays. The results demonstrated that treatment with fursultiamine increased the expression of pro-apoptotic factors such as Bad, Bax, cleaved caspase 3, Fas/CD95, and FADD. Further, fursultiamine increased expression of the tumor suppressor genes p53, p27, and p21. We next planned to examine this vitamin B1 derivative's effect in preventing cancer growth in nude mice xenograft models. In conclusion, our in vitro results indicate that fursultiamine, a vitamin B1 derivative, prevents colon cancer growth through increased regulation of pro-apoptotic pathways and tumor suppressor proteins and thus has the potential for further development as a chemopreventative agent. Citation Format: Sudeep Poludasu, Christian Pompoco, Kota Ramana. Vitamin B1 derivative, fursultiamine, prevents colon cancer growth [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 3178.

Abstract 2653: Integrative analysis of proteomic alterations in human PBMC’s treated by HPK1 inhibitor FB849 reveals enriched pathways from various immune cells leading to potential novel biomarker discovery

Abstract HPK1, also known as MAP4K1, is a critical signaling kinase molecule as a negative feedback loop in various immune cells. Despite the advancement of multiple clinical programs for therapeutic inhibition of HPK1, there is an urgent need to understand HPK1 biology in genome-wide signaling networks not only from lymphocytes but also from monocytes. FB849 is designed as a potent and highly selective inhibitor of HPK1 and is currently in phase I clinical trial with advanced cancer patients (NCT05761223). We aim to generate proteomic and phosphoproteomic profiles to reveal modes of action of FB849 in the broad spectrum of cancer immunity. Human PBMCs were treated with FB849 under conditions with or without CD3/CD28 stimulation. Utilizing an antibody array comprising 1,930 antibodies, the expression of 1,438 distinct proteins was assessed. The antibody arrays in human PBMCs identified a total of 85 and 33 phospho-proteins showing differential expression in response to FB849 treatment compared to the control in the presence or absence of CD3/CD28 stimulations, respectively (fold change &amp;gt; 2, adj. p-value &amp;lt; 0.01). KEGG pathway enrichment analysis demonstrated that the differentially expressed proteins by FB849 in the presence of CD3/CD28 stimulation were significantly enriched in immune-related biological processes, including 'pathways in cancer', 'PI3K-Akt signaling pathway', 'Focal adhesion', 'cytokine-cytokine receptor interaction', 'MAPK signaling pathway', 'JAK-STAT signaling pathway', and 'Th1 and Th2 cell differentiation'. Reactome biology pathway analysis also showed significant enrichment in ‘immune system’, ‘homeostasis’, and ‘cytokine signaling in immune cells’. In addition, we performed LC-MS/MS-based phosphoproteomics in PBMC samples pooled from CT26 mouse syngeneic models treated with 25mg/kg and 50mg/kg of FB849 and its derivative. The proteomics identified 1239 unique phosphorylated proteins and 915 distinct phosphorylation proteins, while 68 and 39 phosphorylated proteins were identified as differentially expressed proteins in FB849 or its derivative treated groups compared with control groups, respectively. The several phosphoproteins exhibited consistent decreases or increases in both FB849 and its derivative. Together, this work reveals genome-wide alterations in the proteomic and phosphoproteomic landscape by FB849 in both mouse PBMC’s and human PBMC’s with clear pathway enrichment potentially from various immune cell lineages. Further exploration of the differential expression of each protein and/or phosphoproteins especially in T cell-, B cell-, and monocyte-enriched biomarkers which link to HPK1-relevant signal transduction is needed to find out their clinical implication and to highlight new understanding of HPK1 signaling network in cancer immunity. Citation Format: Seungmook Lim, Sooryeonhwa Jin, A Yeong Park, Hyekyoung Kim, Seongkon Kim, Jamie Jae Eun Kim, Jinhwa Lee. Integrative analysis of proteomic alterations in human PBMC’s treated by HPK1 inhibitor FB849 reveals enriched pathways from various immune cells leading to potential novel biomarker discovery [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 2653.

Abstract 7303: The algal carotenoid diatoxanthin shows anti-cancer and cardio-protective properties: RNA seq analysis of potential targets

Abstract Introduction: Natural bioactive pigments are of great interest for human health. Marine organisms are underexplored as source of useful metabolites. Diatoms produce diatoxantin (diatox), a xanthophyll with several properties (chemo-preventive, anti-inflammatory, antioxidant, photoprotective, immunomodulatory), that may have beneficial effects. Doxorubicin (doxo) is still one of the most effective chemotherapeutic drugs used in breast cancer (BC) treatment, but has side effects, particularly cardiotoxicity, mainly due to oxidative stress and cardiomyocyte damage. We have assessed the anti-cancer effects of diato, also verifying the absence of cardiotoxicity. Experimental procedures: Diatox was administered to MDA-MB-231 triple negative BC (TNBC) in vitro, in combination with doxo by MTT and by 3D-spheroid assays. The modulation of diatox on the secretome of both TNBC cells and Human Umbilical-Vein Endothelial cell line (HUVEc) was assessed in vitro by angiogenesis arrays. Gene expression analysis was evaluated by measuring different inflammation and angiogenesis gene expression in cells treated with diato, by qPCR; while transcriptomic analysis was performed by RNA-Seq to verify diato effects on pathways involved in TNBC progression and in HUVEc inflammation. Results: Diatox showed an inhibitory effect on the proliferation of MDA-MB-231 cells and, in combination with doxo, was able to inhibit TNBC spheroid growth, reducing their 3D-integrity and decreasing their viability. Diatoxx was able to down-regulate genes involved in inflammatory and angiogenic processes in HUVEc stimulated by TNFα (angiostatin, IL2, IL4 and PECAM-1 in antibody array and TGFβ1, TGFβ2, TIMP1 and TIMP2 gene in qPCR analysis). Moreover, diatox reduced several biological processes involved in inflammation and tumour development and metastasis, as demonstrated by RNA-Seq data. In TNBC cells diatox down-regulated ANGPT1, GPR37, ANG, QRICH2, MUC1 genes involved in angiogenesis processes, while EPHA7 was up-regulated. In TNFα-stimulated HUVEc, diatox down-regulated angiogenin, a powerful inducer of angiogenesis, and HSPA12A and EDNRB, which are involved in the regulation of macrophage cytokine production. Conclusions: Diatox shows to be a promising chemo-preventive and angiopreventive natural compound in cancer prevention and therapy. Microalgae derived drugs can represent a potential and relevant source of novel nutraceutical and preventive substances, which can be considered as supplements in therapeutic strategies for cancer prevention. Acknowledgements: The studies were supported by Stazione Zoologica Anton Dohrn, by "Antitumor Drugs and Vaccines from the Sea "ADViSE" project (PG/2018/0494374). Citation Format: Douglas M. Noonan, Luana Calabrone, Luigi Pistelli, Christophe Brunet, Clementina Sansone, Danilo Morelli, Giovanna Chiorino, Francesca Guana, Adriana Albini. The algal carotenoid diatoxanthin shows anti-cancer and cardio-protective properties: RNA seq analysis of potential targets [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 7303.

Abstract 6951: The role of estrogen-dependent activation of p38 MAPK signaling pathway in inflammatory breast cancer

Abstract Inflammatory breast cancer (IBC) is a rare and aggressive type of breast cancer in which the lymphatic vessels become blocked by cancer cells in the breast tissue. The five-year survival rate for this type of cancer is around 40%, one of the worst among breast cancer. Most IBCs are estrogen receptor (ER) and progesterone receptor (PR) negative and HER2 positive, with some presenting an amplification of HER2 while others are ER, PR, and HER2 negative, formally known as the triple-negative (TNBC) subtype. However, it has been reported that treatment with 17β-Estradiol (E2) of ER-negative IBC and non-IBC cells can exert rapid non-genomic signaling mediated by ERα-36, GPR30, and/or estrogen receptor beta, activating MAPK pathways involved in oncogenic phenotypes. Previous data generated in our lab showed that when cells were treated with estradiol it led to increased phosphorylation levels of p38. However, the specific role p38 MAPK signaling takes on in IBC is unknown. Thus, this study centers on examining the role of estradiol and p38 regulation in the triple-negative IBC cell line model, SUM149PT. We hypothesize that upon estradiol treatment p38 signaling pathway is activated resulting in the over-expression of pro-inflammatory cytokines which regulate pro-tumorigenic phenotypes including the growth and survival of IBC cells. Inflammatory cytokines can promote pro-oncogenic features by promoting cell proliferation, angiogenesis, and immune suppression. We treated SUM149 IBC cells with 10nM of estradiol and measured the levels of human cytokines using a Human Cytokine Antibody Array. In addition, treatment using SB202190, a potent p38 inhibitor, caused a significant decrease in cell viability of several breast cancer cell lines in a dose-dependent manner. Ongoing studies include assessing the effects of p38 inhibition proliferation, invasion, and migration. In summary, a better characterization of the effect of E2 and p38 activation in ER-negative cell lines may provide great insight into novel targeted therapies for IBC. Citation Format: Keyla V. Ramirez, Adrian D. Bonilla-González, Jeisac Guzman-Rivera, Esther A. Peterson-Peguero. The role of estrogen-dependent activation of p38 MAPK signaling pathway in inflammatory breast cancer [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 6951.

Abstract 6809: Tumor-associated macrophages promote bladder cancer cell migration and invasion through activation of CCL20-CCR6 axis

Abstract We focused on Tumor-Associated Macrophages (TAM) and explored its effects on bladder cancer (BC). Co-culturing PMA-treated THP-1 with BC cell lines (UMUC3 and T24) induced THP-1 to develop suppressed CD68 and increased CD206 expression which differentiated into M2-like macrophages. Co-culture of BC with TAM of this M2-like macrophage enhanced the migration and invasive activities of BC, as well as the expression of EMT marker. Human cytokine antibody array of conditioned medium from the co-culture of BC and TAM showed high CCL20, CCL2 and CXCL7. qPCR revealed that these chemokines were mainly derived from TAMs, not BC. Only CCL20 enhanced the migration and invasion of BC and the expression of EMT markers when each of these three recombinant chemokines was administered to BC. Furthermore, inhibiting CCR6, a receptor specific for CCL20, suppressed migration and invasion as expected. In BC, TAMs promote progression and metastasis by secreting CCL20, and inhibition of CCR6 may be one potential therapeutic approach to suppress BC activity. Citation Format: Ryunosuke Nakagawa, Izumi Kouji, Ren Toriumi, Shuhei Aoyama, Taiki Kamjima, Hiroshi Kano, Tomoyuki Makino, Renato Naito, Hiroshi Yaegashi, Takahiro Nohara, Hiroki Nakata, Atsushi Mizokami. Tumor-associated macrophages promote bladder cancer cell migration and invasion through activation of CCL20-CCR6 axis [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 6809.