Murine Cells Research Articles

PARP inhibition leads to synthetic lethality with key splicing-factor mutations in myelodysplastic syndromes.

Splicing factors are frequently mutated in patients with myelodysplastic syndromes and acute myeloid leukaemia. Recent studies have revealed convergent molecular defects caused by splicing factor mutations, among which R-loop dysregulation and resultant genome instability are suggested as contributing factors to disease progression. On the other hand, understanding how mutant cells survive upon aberrant R-loop formation and genome instability is essential for developing novel therapeutics. The immunoprecipitation was performed to identify R-loops in association with PARP1/poly-ADP-ribosylation. The western blot, immunofluorescence, and flow cytometry assays were used to test the cell viability, cell cycle arrest, apoptosis, and ATM activation in mutant cells following the treatment of the PARP inhibitor. The Srsf2(P95H) knock-in murine hematopoietic cells and MLL-AF9 transformed leukaemia model were generated to investigate the potential of the PARP inhibitor as a therapy for haematological malignancies. The disease-causing mutations in SRSF2 activate PARP and elevate the overall poly-ADP-ribosylation levels of proteins in response to R-loop dysregulation. In accordance, mutant cells are more vulnerable to the PARP inhibitors in comparison to the wild-type counterpart. Notably, the synthetic lethality was further validated in the Srsf2(P95H) knock-in murine hematopoietic cell and MLL-AF9 leukaemia model. Our findings suggest that mutant cells antagonise the genome threat caused by R-loop disruption by PARP activation, thus making PARP targeting a promising therapeutic strategy for myeloid cancers with mutations in SRSF2.

Discovery and Optimization of N-Arylated Tetracyclic Dicarboximides That Target Primary Glioma Stem-like Cells.

We recently discovered a novel N-aryl tetracyclic dicarboximide MM0299 (1) with robust activity against glioma stem-like cells that potently and selectively inhibits lanosterol synthase leading to the accumulation of the toxic shunt metabolite 24(S),25-epoxycholesterol. Herein, we delineate a systematic and comprehensive SAR study that explores the structural space surrounding the N-aryl tetracyclic dicarboximide scaffold. A series of 100 analogs were synthesized and evaluated for activity against the murine glioma stem-like cell line Mut6 and for metabolic stability in mouse liver S9 fractions. This study led to several analogs with single-digit nanomolar activity in Mut6 glioblastoma cells that were metabolically stable in S9 fractions. In vivo pharmacokinetic analysis of selected analogs identified compound 52a (IC50 = 63 nM; S9 T1/2 > 240 min) which was orally available (39% plasma; 58% brain) and displayed excellent brain exposure. Chronic oral dosing of 52a during a 2-week tolerability study indicated no adverse effect on body weight nor signs of hematologic, liver, or kidney toxicity.

Dual-Emissive Detection of ATP and Hypochlorite Ions for Monitoring Inflammation-Driven Liver Injury In Vitro and In Vivo.

Reactive oxygen species play a pivotal role in liver disease, contributing to severe liver damage and chronic inflammation. In liver injury driven by inflammation, adenosine-5'-triphosphate (ATP) and hypochlorite ion (ClO-) emerge as novel biomarkers, reflecting mitochondrial dysfunction and amplified oxidative stress, respectively. However, the dynamic fluctuations of ATP and ClO- in hepatocytes and mouse livers remain unclear, and multidetection techniques for these biomarkers are yet to be developed. This study presents RATP-NClO, a dual-channel fluorescent bioprobe capable of synchronously detecting ATP and ClO- ions. RATP-NClO exhibits excellent selectivity and sensitivity for ATP and ClO- ions, demonstrating a dual-channel fluorescence response in a murine hepatocyte cell line. Upon intravenous administration, RATP-NClO reveals synchronized ATP depletion and ClO- amplification in the livers of mice with experimental metabolic dysfunction-associated steatohepatitis (MASH). Through a comprehensive analysis of the principal mechanism of the developed bioprobe and the verification of its reliable detection ability in both in vitro and in vivo settings, we propose it as a unique tool for monitoring changes in intracellular ATP and ClO- level. These findings underscore its potential for practical image-based monitoring and functional phenotyping of MASH pathogenesis.

Non-clinical investigations about cytotoxic and anti-platelet activities of gamma-terpinene.

Gamma-terpinene (γ-TPN) is a cyclohexane monoterpene isolated from plant essential oils, such as tea tree (Melaleuca alternifolia), oregano (Origanum vulgare), rosemary (Rosmarinus officinalis L.), thyme (Thymus vulgaris Marchand), and eucalyptus (Eucalyptus sp.). Terpenes are widely studied molecules pharmacologically active on the cardiovascular system, hemostasis, and antioxidant actions. Herein, it was investigated the cytotoxic and antiplatelet activity of γ-TPN using different non-clinical laboratory models. For in silico evaluation, the PreADMET, SwissADME, and SwissTargetPrediction softwares were used. Molecular docking was performed using the AutoDockVina and BIOVIA Discovery Studio databases. The cytotoxicity of γ-TPN was analyzed by the MTT assay upon normal murine endothelial SVEC4-10 and fibroblast L-929 cells. Platelet aggregation was evaluated with platelet-rich (PRP) and platelet-poor (PPP) plasma from spontaneously hypertensive rats (SHR), in addition to SVEC4-10 cells pre-incubated with γ-TPN (50, 100, and 200 µM) for 24 h. SHR animals were pre-treated by gavage with γ-TPN for 7 days and divided into four groups (negative control, 25, 50, and 100 mg/kg). Blood samples were collected to measure nitrite using the Griess reagent. Gamma-TPN proved to be quite lipid-soluble (Log P = +4.50), with a qualified profile of similarity to the drug, good bioavailability, and adequate pharmacokinetics. It exhibited affinity mainly for the P2Y12 receptor (6.450 ± 0.232 Kcal/mol), moderate cytotoxicity for L-929 (CC50 = 333.3 µM) and SVEC 4-10 (CC50 = 366.7 µM)cells. The presence of γ-TPN in SVEC 4-10 cells was also able to reduce platelet aggregation by 51.57 and 44.20% at lower concentrations (50 and 100 µM, respectively). Then, γ-TPN has good affinity with purinergic receptors and an effect on the reversal of platelet aggregation and oxidative stress, being promising and safe for therapeutic targets and subsequent studies on the control of thromboembolic diseases.

Cardiac differentiation is modulated by anti-apoptotic signals in murine embryonic stem cells

BACKGROUND Embryonic stem cells (ESCs) serve as a crucial ex vivo model, representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos. ESCs exhibit a unique combination of self-renewal potency, unlimited proliferation, and pluripotency. The latter is evident by the ability of the isolated cells to differentiate spontaneously into multiple cell lineages, representing the three primary embryonic germ layers. Multiple regulatory networks guide ESCs, directing their self-renewal and lineage-specific differentiation. Apoptosis, or programmed cell death, emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development. However, the molecular mechanisms underlying the dynamic interplay between differentiation and apoptosis remain poorly understood. AIM To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells, using mouse ESC (mESC) models - mESC-B-cell lymphoma 2 (BCL-2), mESC-PIM-2, and mESC-metallothionein-1 (MET-1) - which overexpress the anti-apoptotic genes Bcl-2 , Pim-2 , and Met-1 , respectively. METHODS mESC-T2 (wild-type), mESC-BCL-2, mESC-PIM-2, and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation. The hanging drop method was adopted to generate embryoid bodies (EBs) and induce terminal differentiation of mESCs. The size of the generated EBs was measured in each condition compared to the wild type. At the functional level, the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control. At the molecular level, quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers: Troponin T, GATA4, and NKX2.5. Additionally, troponin T protein expression was evaluated through immunofluorescence and western blot assays. RESULTS Our findings showed that the upregulation of Bcl-2 , Pim-2 , and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs, in comparison with their wild-type counterpart. Additionally, a decrease in the count of beating cardiomyocytes among differentiated cells was observed. Furthermore, the mRNA expression of three cardiac markers - troponin T, GATA4, and NKX2.5 - was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line. Moreover, the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression. CONCLUSION Our findings revealed that the upregulation of Bcl-2 , Pim-2 , and Met-1 genes altered cardiac differentiation, providing insight into the intricate interplay between apoptosis and ESC fate determination.

Mitotic MTH1 inhibitor TH1579 induces PD-L1 expression and inflammatory response through the cGAS-STING pathway

The mitotic MTH1 inhibitor TH1579 is a dual inhibitor that inhibits mitosis and incorporation of oxidative DNA damage and leads to cancer-specific cell death. The response to immune checkpoint inhibitor (ICI) treatment is often augmented by DNA damaging agents through the cGAS-STING pathway. This study investigates whether TH1579 can improve the efficacy of immune checkpoint blockades through its immunomodulatory properties. Various human and murine cancer cell lines were treated with mitotic MTH1i TH1579, and the expression of PD-L1 and T-cell infiltration-related chemokines was analysed by flow cytometry and real-time qPCR. Syngeneic mouse models were established to examine the combined effect of TH1579 and PD-L1 blockade. In our investigation, we found that TH1579 upregulates PD-L1 expression at both the protein and mRNA levels in human cancer cell lines. However, in murine cell lines, the increase was less pronounced. An in vivo experiment in a syngeneic mouse melanoma model showed that TH1579 treatment significantly increased the efficacy of atezolizumab, an anti-PD-L1 antibody, compared to vehicle or atezolizumab monotherapy. Furthermore, TH1579 exhibited immune-modulatory properties, elevating cytokines such as IFN-β and chemokines including CCL5 and CXCL10, in a cGAS-STING pathway-dependent manner. In conclusion, TH1579 has the potential to improve ICI treatment by modulating immune checkpoint-related proteins and pathways.

Mechanically suitable and osteoinductive 3D-printed composite scaffolds with hydroxyapatite nanoparticles having diverse morphologies for bone tissue engineering.

The challenge of integrating hydroxyapatite nanoparticles (nHAp) with polymers is hindered by the conflict between the hydrophilic and hygroscopic properties of nHAp and the hydrophobic properties of polymers. This conflict particularly affects the materials when calcium phosphates, including nHAp, are used as a filler in composites in thermal processing applications such as 3D printing with fused filament fabrication (FFF). To overcome this, we propose a one-step surface modification of nHAp with calcium stearate monolayer. Moreover, to build the scaffold with suitable mechanical strength, we tested the addition of nHAp with diverse morphology-spherical, plate- and rod-like nanoparticles. Our analysis showed that the composite of polycaprolactone (PCL) reinforced with nHAp with rod and plate morphologies modified with calcium stearate monolayer exhibited a significant increase in compressive strength. However, composites with spherical nHAp added to PCL showed a significant reduction in compressive modulus and compressive strength, but both parameters were within the applicability range of hard tissue scaffolds. None of the tested composite scaffolds showed cytotoxicity in L929 murine fibroblasts or MG-63 human osteoblast-like cells, supporting the proliferation of the latter. Additionally, PCL/nHAp scaffolds reinforced with spherical nHAp caused osteoactivation of bone marrow human mesenchymal stem cells, as indicated by alkaline phosphatase activity and COL1, RUNX2, and BGLAP expression. These results suggest that the calcium stearate monolayer on the surface of the nHAp particles allows the production of polymer/nHAp composites suitable for hard tissue engineering and personalized implant production in 3D printing using the FFF technique.

Maclura cochinchinensis (Lour.) Corner Heartwood Extracts Containing Resveratrol and Oxyresveratrol Inhibit Melanogenesis in B16F10 Melanoma Cells

This study aimed to isolate and purify resveratrol and oxyresveratrol from the heartwoods of Maclura cochinchinensis, and to evaluate their inhibitory effects on melanogenesis in B16F10 murine melanoma cells. A methanol maceration process yielded a crude extract comprising 24.86% of the initial mass, which was subsequently analyzed through HPTLC, HPLC, and LC-MS/MS. These analyses revealed the presence of resveratrol and oxyresveratrol at concentrations of 4.32 mg/g and 33.6 mg/g in the extract, respectively. Initial purification employing food-grade silica gel column chromatography separated the extract into two fractions: FA, exhibiting potent inhibition of both tyrosinase activity and melanogenesis, and FM, showing no such inhibitory activity. Further purification processes led to the isolation of fractions Y11 and Gn12 with enhanced concentrations of resveratrol (94.9 and 110.21 mg/g, respectively) and fractions Gn15 and Gn16 with elevated levels of oxyresveratrol (321.93 and 274.59 mg/g, respectively), all of which significantly reduced melanin synthesis. These outcomes affirm the substantial presence of resveratrol and oxyresveratrol in the heartwood of M. cochinchinensis, indicating their promising role as natural agents for skin lightening.

Limited impact of cancer-derived gangliosides on anti-tumor immunity in colorectal cancer.

Aberrant glycosylation is a key mechanism employed by cancer cells to evade immune surveillance, induce angiogenesis and metastasis, among other hallmarks of cancer. Sialic acids, distinctive terminal glycan structures located on glycoproteins or glycolipids, are prominently upregulated across various tumor types, including colorectal cancer (CRC). Sialylated glycans modulate anti-tumor immune responses through their interactions with Siglecs, a family of glycan-binding receptors with specificity for sialic acid-containing glycoconjugates, often resulting in immunosuppression. In this paper, we investigated the immunomodulatory function of ST3Gal5, a sialyltransferase that catalyzes the addition of α2-3 sialic acids to glycosphingolipids, since lower expression of ST3Gal5 is associated with better survival of CRC patients. We employed CRISPR/Cas9 to knock out the ST3Gal5 gene in two murine CRC cell lines MC38 and CT26. Glycomics analysis confirmed the removal of sialic acids on glycolipids, with no discernible impact on glycoprotein sialylation. Although knocking out ST3Gal5 in both cell lines did not affect in vivo tumor growth, we observed enhanced levels of regulatory T cells in CT26 tumors lacking ST3Gal5. Moreover, we demonstrate that the absence of ST3Gal5 affected size and blood vessel density only in MC38 tumors. In summary, we ascertain that sialylation of glycosphingolipids has a limited influence on the anti-tumor immune response in CRC, despite detecting alterations in the tumor microenvironment, possibly due to a shift in ganglioside abundance.

#2949 Diverse effect of STAT3 inhibition on murine primary tubular epithelial cells

Abstract Background and Aims The incidence of chronic kidney diseases increases worldwide, and kidney fibrosis is the final common pathway regardless of the etiology. TGF-β plays a crucial role in the pathogenesis, affecting pro-fibrotic genes and transcription factors. STAT3 (signal transducer and activator of transcription 3) acts via the JAK/STAT signaling. Inhibition of STAT3 in animal studies was shown to attenuate the progression of kidney fibrosis. Yet, the effect of STAT3 inhibition on renal tubular epithelial cells has not been elucidated. We aimed to investigate in vitro the impact of pharmacological STAT3 inhibition with NSC74859 (NSC) and STATTIC on the expression of fibrosis associated genes of primary tubular epithelial cells. Method Primary tubular epithelial cells from kidneys of C57Bl6/J mice were isolated and used in our studies. Cells (P10-P15) were seeded on gelatin-coated 6-well culture plates (100000 cells/well) and subjected to treatment with 10 ng/ml TGF-β, 100 µM NSC, 10 µM STATTIC or vehicle (n = 3/group) as follows: Control; NSC; STATTIC; TGF-β; TGF-β+NSC; TGF-β+STATTIC. After 24 h treatment, mRNA and protein expressions were analyzed. Statistical evaluation was performed using Kruskal-Wallis test. Results In cells not treated with TGF-β, neither NSC nor STATTIC treatment affected the expression of Tgfb1 (TGF-β) and Acta2 (SMA) genes compared to controls (100%), but reduced Col1a1 expression to 58% and 54%, respectively (p < 0.05). NSC reduced to 54%, while STATTIC tended to increase (169%) the gene expression of Egr2 transcription factor. TGF-β treatment induced STAT3 phosphorylation by 45% accompanied by a fibrotic response and epithelial-to-mesenchymal transition (EMT) as indicated by significantly increased Tgfb1, Acta2, Egr2 and Col1a1 (p < 0.05). NSC treatment did not inhibit the TGF-β-induced Tgfb1 upregulation (178%), but markedly, although not significantly, reduced the expression of Acta2 (100%), Egr2 (98%) and Col1a1 (114%) in the TGF-β+NSC group. Interestingly, although STATTIC treatment dramatically inhibited STAT3 expression (38%, p < 0.05) it significantly enhanced the TGF-β-induced overexpression of Tgfb1 (210%, p < 0.01) and Egr2 (307%, p < 0.05), while markedly reduced Acta2 (64%, p < 0.001) and Col1a1 (81%, p < 0.01) in the TGF-β+STATTIC group. Conclusion Our results indicate that in vitro inhibition of STAT3 using two inhibitors with same mode of action lead to diverse responses in primary murine tubular epithelial cells, partially suppressing EMT markers but inducing pro-fibrotic EGR2. Further studies are needed to clarify the underlying mechanisms.

#1995 Runt-related Transcription Factor 1 (RUNX1) is a mediator of acute kidney injury

Abstract Background and Aims A better understanding of the pathogenesis of acute kidney injury (AKI) may lead to new therapeutic approaches. Kidney transcriptomics analyses of murine folic acid-induced AKI (FA-AKI) identified Runx1 as the most upregulated RUNX family gene. RUNX1 is a key regulator of hematopoiesis, associated to leukemias and other non-immunological cancers and modulates myoblast proliferation during muscle regeneration and cardiac remodelling after myocardial infarction. In kidney diseases, RUNX1 is overexpressed in polycystic kidney disease and kidney cancer and favours fibrosis. Method We examined the expression of RUNX1 in folic acid-AKI (FA-AKI), in bacterial lipopolysaccharide (LPS)-induced cytokine storm-AKI (CS-AKI) and in human AKI. For this, female 12- to 14-week-old C57BL/6J wild type (WT) mice, received a single intraperitoneal (i.p) injection of acid folic (250 mg/kg), LPS (5 mg/kg) or vehicle. In cultured murine tubular MCT cells, we explored the expression and role of RUNX1 in response to the cytokine TWEAK or LPS. The chemical inhibitor of RUNX1 Ro5-3335 and a specific small interfering RNA (siRNA) against RUNX1 were used in cultured cells to explore the role of RUNX1 in TWEAK and LPS-induced inflammation. Ro5-3335 was also used in murine AKI (FA-AKI and CS-AKI) to test its potential as a therapeutic target. In all experimental models, plasma samples were collected to assess renal function. Kidneys were collected for RNA (RT-PCR), protein (Western blot, ELISA, immunohistochemistry) and histopathologic studies. Results Kidney transcriptomics identified Runx1 as the most upregulated Runx gene in FA-AKI at 24 h and the most overactive transcription factor in upstream regulator analysis. RUNX1 overexpression in FA-AKI was validated at mRNA and protein levels and localized mainly to tubular cell nuclei. CS-AKI also upregulated kidney RUNX1 in nuclei. In a transcriptomics array of murine tubular cells stimulated with TWEAK for 6 h, Runx1 was upregulated compared with non-stimulated cells, while Runx2 and Runx3 were not. RT-PCR confirmed an early upregulation of Runx1 (3 h) followed by a progressive return to baseline by 24 h. TWEAK also increased total and nuclear RUNX1 protein levels from 6 h onwards. Since kidney RUNX1 is also increased in CS-AKI, we tested whether LPS regulated RUNX1 in cultured tubular cells. LPS transiently increased Runx1 mRNA expression and it also increased whole cell and nuclear RUNX1 protein levels. Mechanistically, RUNX1 bound to the Il-6 gene promoter in response to TWEAK and LPS, and RUNX1 targeting with the chemical inhibitor Ro5-3335 or a specific siRNA prevented the TWEAK- and LPS-induced upregulation of IL-6. NFκB1 p50 pathway is involved in the RUNX1/IL-6 axis since the specific p50 inhibitor SN50 decreased the nuclear accumulation of RUNX1 and IL-6 expression in TWEAK- or LPS-stimulated MCT cells. In vivo, preventive Ro5-3335 improved kidney function and reduced inflammation in FA-AKI and CS-AKI. Moreover, in FA-AKI, Ro5-3335 also prevented the expression of profibrotic genes. However, Ro5-3335 administration after the insult only improved kidney function in CS-AKI. Kidney transcriptomics identified inflammatory genes and transcription factors such as Yap1 and p53 as key targets of Ro5-3335 in CS-AKI. Conclusion RUNX1 contributes to AKI by driving the expression of genes involved in inflammation and represents a novel therapeutic target in AKI.

#2711 Cardiomyocytes exposed to medium conditioned by kidney cells treated with the uremic toxin p-cresyl sulfate undergo changes in cell viability

Abstract Background and Aims Cardiorenal Syndrome (CRS) is a condition involving the heart and the kidneys, where a disturbance in one of these organs can lead to alterations in the other. High levels of uremic toxins (UTs) such as p-Cresyl Sulfate (PCS) can induce renal injury. Furthermore, UTs play a crucial role in inflammatory status and cardiac function. Therefore, it is crucial that studies regarding the influence of uremic toxins in the context of cardiorenal syndrome be conducted. Nevertheless, few studies have been conducted in the context of Acute Kidney Injury (AKI) causing acute damage to the heart, and few mechanisms are still known. The objective of the study was to evaluate the role of PCS in cell viability and inflammatory genes in renal and cardiac cells, to simulate cardiorenal syndrome due to excessive PCS. Method The HEK293 (human epithelial renal cells) and H9c2 (murine cardiomyocyte cells) cell lines were used. HEK293 was treated with concentrations of 0.25 mM, 0.5 mM, 1 mM, and 1.5 mM PCS for 6, 24, and 48 hours. H9c2 was treated with PCS (1.5 mM) and conditioned media from PCS-treated renal cells (1.5 mM) for 6 and 24 hours. MTT assays, growth curve analysis, and Hoechst-PI staining were performed. Gene expression was evaluated by RT-qPCR. ANOVA followed by Bonferroni post-test was conducted, considering P < 0.05 as significant. Results PCS treatment led to a time and dose-dependent decrease in cell viability. The cell growth curve indicated a negative impact across time on renal cell proliferation at all different PCS concentrations. Hoechst-PI assay revealed increased cell death in the treatment with PCS (1.5 mM) for 24 hours. There was no change in the gene expression of IL-6, IL-1β, Bax, and Bcl-2 in renal cells. Cardiac cell viability decreased both after direct PCS treatment (85.3%) and exposure to PCS-conditioned media from renal cells for 24 hours (87.3%). Conclusion PCS reduces renal cell viability and inhibits cell proliferation without affecting the inflammatory status and apoptosis signaling within the evaluated time frames. Cardiac cell viability decreased after PCS treatment, either directly or indirectly, suggesting a cardiotoxic effect of PCS, independent of changes in renal cells.

#2222 Suppression of miR-199a-3p ameliorates TGF-β-induced EMT in renal tubular cells

Abstract Background and Aims Various kidney conditions, such as acute kidney injury and fibrosis, cause dysregulation of miRNAs. TGF-β is one of the most potent inducer of epithelial-to-mesenchymal transition (EMT) and fibrosis. We recently reported increased miR-199 expression in TGF-β-induced kidney fibrosis mouse model (doi: 10.3390/ijms242115520). However, the exact role of miR-199 in fibrosis and EMT development is still unknown. Method Primary tubular cells were isolated from a 4-week-old C57BL6 mouse and kept in DMEM/F12 medium containing 2% serum, supplemented with insulin/transferrin/selenium. Cells were treated for 24 hours with 30 nM miR-199-3p miRNA inhibitor (anti-miR), with or without TGF-β (10 ng/ml). The expression of profibrotic genes, as well as miR-199a-3p, was assessed by qPCR. Western blot was used to evaluate the protein expression. Results are presented as mean fold expression relative to calibrator. Statistical significance was assessed using the Kruskal-Wallis test, and the level of significance was set to p < 0.05. Results The miR-199a-3p expression in the anti-miR transfected control and TGF-β groups decreased by 93.5% and 85.5%, respectively, as compared to non-transfected groups (p < 0.05). Morphologically, cells transfected with miR-199a-3p and TGF-β1 showed no difference from cells exposed to only TGF-β1 but appeared more elongated compared to the control groups. However, the suppression of miR-199a-3p alleviated TGF-β1-induced fibrotic cell responses as shown by downregulated fibronectin mRNA (Fn1) and protein expression by 30.2% (p < 0.05) and by 4.3%, respectively. This was accompanied by reduced Acta2 (α-SMA) and Tgfb1 mRNA expressions by 30.2% and 17.5%, respectively (p < 0.05). Conclusion Our findings suggest that in murine renal tubular epithelial cells, miR-199a-3p suppresses TGF-β1-induced EMT by inhibiting FN1, Acta2, and Tgfb1 both at mRNA and protein levels. Further studies are needed to elucidate the underlying molecular interactions.

#2485 CellMatchR—RNA-seq based matching of kidney cell lines to kidney reference cell types

Abstract Background and Aims Cultured kidney cell lines are broadly used to model the physiology and pathophysiology of the kidney. Due to immortalization, passaging and culture conditions, a cell line's gene expression profile might differ greatly from the primary cells it was originally derived from. This might be further enhanced by experimental treatments with compounds or genetic modifications. It is therefore a relevant question if a given cell culture system is still the appropriate model for a certain disease or scientific investigations. Reasoning that RNA sequencing (RNA-seq) based transcriptomes are generated as part of many experimental setups and hence may be used to address this question, we developed and tested an RNA-seq based approach, CellMatchR. This approach matches kidney cell lines, primary cells and even tissue specimen to known kidney reference cell types to determine which primary kidney cell type is the most similar and to what degree global gene expression or the expression of selected marker genes differs. Method CellMatchR uses published murine and human kidney single cell- and tubule-level transcriptomic datasets from healthy mice and human donors as references. Single cell datasets were further processed to pseudobulk references for each of the contained cell types. Then RNA-seq data from cell lines or tissues of interest (test data) was compared to the reference (pseudo)bulk data with Spearman correlations of gene counts-per-million values (CPM) or Euclidean distance using log-transformed CPM values. Both approaches were systematically tested for various combinations of test and reference datasets across different species and using global gene expression (i.e. all genes measured in reference and test datasets) and a set of 315 manually curated, kidney cell type marker genes. Results We sequentially used different kidney tissue types, primary cells and cell lines as positive controls and matched them to our reference datasets. Spearman correlations of gene expression rankings showed the highest correlation coefficients with biologically correct reference cell types (examples in Fig. 1A-C). We found Spearman correlations to be superior to Euclidean distances as method of comparison. Analyses based on global gene expression compared to our curated set of 315 kidney cell type marker genes yielded similar results. Notably, correlation coefficients were higher and showed less variation between reference cell types when using the global gene set. Matchings across species, i.e. using murine test and human reference data or vice versa still yielded mostly correct results. However, correlation coefficients were generally lower (i.e. rho = 0.9 vs. 0.6) and varied more across reference datasets if comparisons were performed across species. Using published RNA-seq data for different cell lines of the proximal tubule (e.g. human HK-2 and opossum OKH cells) we observed low similarities with proximal tubule cells (Fig. 1D), whereas two tested cell lines of the collecting duct (mIMCD-3 and mpkCCD cells) plausibly showed the highest similarity to medullary cell types like collecting duct and Loop of Henle cells. The most similar reference cell types did not change in mIMCD-3 cells when kept in 2-dimensional vs. 3-dimensional culture conditions, and in mpkCCD cells when the osmolality of the culture medium was changed from 300 to 600 mosmol/kg. Also, a Pkd1 knockout in mIMCD-3 cells did not change the most similar reference cell type in our analyses (Fig. 1E and F). Conclusion Our CellMatchR approach uses publicly available kidney single cell and bulk RNA-seq datasets and combines these with simple, computationally fast yet effective statistical methods to determine similarities of kidney cell lines and tissue samples to reference cell types of interest. It can easily be implemented by trained users or integrated into online resources for usage with a visual interface. It relies on RNA-seq data from the cell lines of interest and hence presents a feasible complementary method to check the general similarity of cell culture models to kidney cell types and assess their stability across experimental conditions.

#984 Macrophage BRCC3 ameliorates sepsis-induced kidney injury by mediating renal NLRP3 inflammation

Abstract Background and Aims Sepsis-induced kidney injury (S-AKI) is an inflammatory disease characterized by high mortality rates. Macrophages are key inflammatory cells in the pathogenesis of S-AKI, and a rationale to target macrophage-endothelial cell interactions has been elucidated. Some study reported that BRCC3, a deubiquitylating enzyme, which is a critical regulator of NLRP3 activity in vitro experiments. However, the role of Macrophage BRCC3 role in S-AKI remains unclear. Method The S-AKI model was established by injecting lipopolysaccharide (LPS) into wild-type (WT) and macrophage-specific BRCC3 knockdown (KO-Mφ) mice. Transfecting RAW264.7 macrophages with siBRCC3 and establishing the co-culture system of RAW264.7 macrophages and murine tubular epithelial cells (mTECs) in Transwell. Results We found that macrophage BRCC3 deficiency ameliorates S-AKI. Compared with WT-S-AKI mice, the macrophage infiltration was decreased, the serum creatinine, BUN and the renal histologic injury scoring was decreased, as well as the expression of NLRP3 and Pro-Caspase-1 was clearly attenuated in KO-Mφ S-AKI mice. However, the serum IL-1β, IL-6, and TNF-α changes had on different between S-AKI groups. In vitro study, knockdown of BRCC3 in macrophages decreased NLRP3 inflammasome activation and cell apoptosis in mTECs. Mechanistically, BRCC3 knockdown promoted K63-linked ubiquitination of NLRP3 in macrophages. NLRP3 overexpression in mTEC abolished the renal protective effects of macrophages BRCC3 knockoff in S-AKI mice. Conclusion We showed that the macrophage BRCC3 as a key regulator in the activation of NLRP3 inflammasome by ubiquitination pathway in S-AKI. Our study unveils that macrophage BRCC3 has the potential to be the novel therapeutic target for S-AKI treatment.

Mechanisms of Gelofusine Protection against Polymyxin B-associated Renal Injury.

Polymyxins are a last-resort treatment option for multidrug-resistant Gram-negative bacterial infections, but they are associated with nephrotoxicity. Gelofusine was previously shown to reduce polymyxin-associated kidney injury in an animal model. However, the mechanism(s) of renal protection has not been fully elucidated. Here, we report the use of a cell culture model to provide insights into the mechanisms of renal protection. Murine epithelial proximal tubular cells were exposed to polymyxin B. Cell viability, polymyxin B uptake, mitochondrial superoxide production, nuclear morphology, and apoptosis activation were evaluated with or without concomitant gelofusine. A megalin-knockout cell line was used as an uptake inhibition control. Methionine was included in selected experiments as an antioxidant control. A polymyxin B concentration-dependent reduction in cell viability was observed. Increased viability was observed in megalin-knockout cells following comparable polymyxin B exposures. Compared to polymyxin B exposure alone, concomitant gelofusine and methionine significantly increased cell viability, reduced mitochondrial superoxide production, and improved nuclear morphology. Gelofusine, but not methionine, significantly reduced polymyxin B uptake and ratio of Bax/Bcl-2 protein (a biomarker of intrinsic apoptosis). Gelofusine and methionine were more effective at reducing renal cell injury in combination than either agent alone. The mechanisms of renal protection by gelofusine involve decreasing cellular drug uptake, reducing subsequent oxidative stress and apoptosis activation. These findings would be valuable for translational research into clinical strategies to attenuate drug-associated acute kidney injury.

Double-faced CX3CL1 enhances lymphangiogenesis-dependent metastasis in an aggressive subclone of oral squamous cell carcinoma.

Because cancer cells have a genetically unstable nature, they give rise to genetically different variant subclones inside a single tumor. Understanding cancer heterogeneity and subclone characteristics is crucial for developing more efficacious therapies. Oral squamous cell carcinoma (OSCC) is characterized by high heterogeneity and plasticity. On the other hand, CX3C motif ligand 1 (CX3CL1) is a double-faced chemokine with anti- and pro-tumor functions. Our study reported that CX3CL1 functioned differently in tumors with different cancer phenotypes, both in vivo and in vitro. Mouse OSCC 1 (MOC1) and MOC2 cells responded similarly to CX3CL1 in vitro. However, in vivo, CX3CL1 increased keratinization in indolent MOC1 cancer, while CX3CL1 promoted cervical lymphatic metastasis in aggressive MOC2 cancer. These outcomes were due to double-faced CX3CL1 effects on different immune microenvironments indolent and aggressive cancer created. Furthermore, we established that CX3CL1 promoted cancer metastasis via the lymphatic pathway by stimulating lymphangiogenesis and transendothelial migration of lymph-circulating tumor cells. CX3CL1 enrichment in lymphatic metastasis tissues was observed in aggressive murine and human cell lines. OSCC patient samples with CX3CL1 enrichment exhibited a strong correlation with lower overall survival rates and higher recurrence and distant metastasis rates. In conclusion, CX3CL1 is a pivotal factor that stimulates the metastasis of aggressive cancer subclones within the heterogeneous tumors to metastasize, and our study demonstrates the prognostic value of CX3CL1 enrichment in long-term monitoring in OSCC.

Bacillus Calmette-Guérin immunotherapy induces an efficient antitumor response to control murine melanoma depending on MyD88 signaling

Bacillus Calmette-Guérin (BCG) is the first line treatment for bladder cancer and it is also proposed for melanoma immunotherapy. BCG modulates the tumor microenvironment (TME) inducing an antitumor effective response, but the immune mechanisms involved still poorly understood. The immune profile of B16-F10 murine melanoma cells was assessed by infecting these cells with BCG or stimulating them with agonists for different innate immune pathways such as TLRs, inflammasome, cGAS-STING and type I IFN. B16-F10 did not respond to any of those stimuli, except for type I IFN agonists, contrasting with bone marrow-derived macrophages (BMDMs) that showed high production of proinflammatory cytokines. Additionally, we confirmed that BCG is able to infect B16-F10, which in turn can activate macrophages and spleen cells from mice in co-culture experiments. Furthermore, we established a subcutaneous B16-F10 melanoma model for intratumoral BCG treatment and compared wild type mice to TLR2-/-, TLR3-/-, TLR4-/-, TLR7-/-, TLR3/7/9-/-, caspase 1-/-, caspase 11-/-, IL-1R-/-, cGAS-/-, STING-/-, IFNAR-/-, MyD88-/-deficient animals. These results in vivo demonstrate that MyD88 signaling is important for BCG immunotherapy to control melanoma in mice. Also, BCG fails to induce cytokine production in the co-culture experiments using B16-F10 and BMDMs or spleen cells derived from MyD88-/- compared to wild-type (WT) animals. Immunotherapy with BCG was not able to induce the recruitment of inflammatory cells in the TME from MyD88-/- mice, impairing tumor control and IFN-γ production by T cells. In conclusion, MyD88 impacts on both innate and adaptive responses to BCG leading to an efficient antitumor response against melanoma.

How Does the Concentration of Technetium-99m Radiolabeled Gold Nanoparticles Affect Their In Vivo Biodistribution?

Gold nanoparticles (AuNPs) radiolabeled with therapeutic and diagnostic radioisotopes have been broadly studied as a promising platform for early diagnosis and treatment of many diseases including cancer. Our main goal for this study was the comparison of the biodistribution profiles of four different concentrations of gold nanoconjugates radiolabeled with Technetium-99m (99mTc). More specifically, AuNPs with an average diameter of 2 nm were functionalized with a tridentate thiol ligand. Four different concentrations were radiolabeled with 99mTc-tricarbonyls with high radiolabeling yields (>85%) and were further purified, leading to radiochemical purity of >95%. In vitro stability of the radiolabeled nanoconstructs was examined in cysteine and histidine solutions as well as in human serum, exhibiting robust radiolabeling up to 24 h post-preparation. Moreover, in vitro cytotoxicity studies were carried out in 4T1 murine mammary cancer cells. In vivo tracking of the radiolabeled nanoconjugates at both concentrations was examined in normal mice in order to examine the effect of AuNPs’ concentration on their in vivo kinetics. Our work demonstrates that varying concentrations of radiolabeled AuNPs lead to notably different biodistribution profiles.

Polysaccharides from a Fermented Beverage Induce Nitric Oxide and Cytokines in Murine Macrophage Cell Line

Polysaccharides from a Fermented Beverage Induce Nitric Oxide and Cytokines in Murine Macrophage Cell Line