Murine Fibroblasts Research Articles

A Promising Approach to Target Colorectal Cancer Using Hybrid Triarylmethanes.

Aiming to create an innovative series of anti-colorectal cancer agents, we designed in this work hybrid triarylmethane compounds. Three hybrid triarylmethanes and their corresponding N-oxide analogues were successfully synthesized using an efficient procedure that involved connecting two triarylmethane molecules, through mono-, bi-, and triethylene glycol fragments. In our pursuit to develop more soluble molecules, we synthesized a hybrid triarylmethane featuring a lysine-based spacer through a convergent strategy involving 7 steps. All hybrid compounds were assessed for their antiproliferative activity on human HT-29 and HCT116 colorectal cancer (CRC) cell lines. Three pyridine N-oxide analogs demonstrated notable antiproliferative potential among the set of tested compounds, with IC50 values ranging from 18 to 24 μM on both human CRC cell lines analyzed. A cytotoxicity study conducted on murine fibroblasts revealed that these three active compounds were not toxic at the IC50 values, indicating their suitability for further drug development. A docking study was conducted on two representative compounds, one for each series and protein kinase B (AKT) was identified as a potential target of their in anti-cancer effects. A computational drug-likeness study predicted favourable oral and intestinal absorption efficiency.

Syntheses, structures and biological activities of new pyridinyl lactones

Several novel lactones containing either a pyridin-3-yl or a pyridin-2-yl ring in their structure were synthesized: trans δ halolactones 6–8a,b (chloro‑, bromo‑ and iodo-), γ iodolactone 11b and isomeric γ hydroxylactones 9–10a,b. Their structures were confirmed by NMR, IR and HR-MS and additionally by single crystal X-ray diffraction for trans δ bromolactone 6a, trans δ chlorolactone 7b and γ hydroxylactone 10b. Spectral analyses were further supported by DFT calculations. In testing of the compounds for antibacterial activity, none of the lactones exhibited activity against several bacteria strains. However, all lactones exhibited significant cytotoxicity against normal eukaryotic murine fibroblast L929 and human gastric adenocarcinoma AGS cell lines with lactones possessing a pyridine-2-yl ring being more cytotoxic than their pyridine-3-yl counterparts.

KDM6A regulates immune response genes in multiple myeloma

AbstractThe histone H3 at lysine 27 (H3K27) demethylase lysine demethylase 6A (KDM6A) is a tumor suppressor in multiple cancers, including multiple myeloma (MM). We created isogenic MM cells disrupted for KDM6A and tagged the endogenous protein to facilitate genome-wide studies. KDM6A binds genes associated with immune recognition and cytokine signaling. Most importantly, KDM6A binds and activates NLRC5 and CIITA, which encode regulators of major histocompatibility complex genes. Patient data indicate that NLRC5 and CIITA are downregulated in MM with low KDM6A expression. Chromatin analysis shows that KDM6A binds poised and active enhancers and KDM6A loss led to decreased H3K27ac at enhancers, increased H3K27me3 levels in body of genes bound by KDM6A, and decreased gene expression. Reestablishing histone acetylation with an HDAC3 inhibitor leads to upregulation of major histocompatibility complex expression, offering a strategy to restore immunogenicity of KDM6A-deficient tumors. Loss of Kdm6a in Kirsten rat sarcoma virus (K-RAS)-transformed murine fibroblasts led to increased growth in vivo associated with decreased T-cell infiltration.

An insight into the role of branched-chain α-keto acid dehydrogenase (BKD) complex in branched-chain fatty acid biosynthesis and virulence of Listeria monocytogenes.

Listeria monocytogenes is a foodborne bacterial pathogen that causes listeriosis. Positive regulatory factor A (PrfA) is a pleiotropic master activator of virulence genes of L. monocytogenes that becomes active upon the entry of the bacterium into the cytosol of infected cells. L. monocytogenes can survive and multiply at low temperatures; this is accomplished through the maintenance of appropriate membrane fluidity via branched-chain fatty acid (BCFA) synthesis. Branched-chain α-keto acid dehydrogenase (BKD), which is composed of four polypeptides encoded by lpd, bkdA1, bkdA2, and bkdB, is known to play a vital role in BCFA biosynthesis. Here, we constructed BKD-deficient Listeria strains by in-frame deletion of lpd, bkdA1, bkdA2, and bkdB genes. To determine the role in in vivo and in vitro, mouse model challenges, plaque assay in murine L2 fibroblast, and intracellular replication in J744A.1 macrophage were conducted. BKD-deficient strains exhibited defects in BCFA composition, virulence, and PrfA-regulon function within the host cells. Transcriptomics analysis revealed that the transcript level of the PrfA-regulon was lower in ΔbkdA1 strain than those in the wild-type. This study demonstrates that L. monocytogenes strains lacking BKD complex components were defective in PrfA-regulon function, and full activation of wild-type prfA may not occur within host cells in the absence of BKD. Further study will investigate the consequences of BKD deletion on PrfA function through altering BCFA catabolism.IMPORTANCEListeria monocytogenes is the causative agent of listeriosis, a disease with a high mortality rate. In this study, we have shown that the deletion of BKD can impact the function of PrfA and the PrfA-regulon. The production of virulence proteins within host cells is necessary for L. monocytogenes to promote its intracellular survival and is likely dependent on membrane integrity. We thus report a link between L. monocytogenes membrane integrity and the function of PrfA. This knowledge will increase our understanding of L. monocytogenes pathogenesis, which may provide insight into the development of antimicrobial agents.

RETRACTION.

S. Mukherjee, S. Manna, D. Pal, P. Mukherjee, C. K. Panda,Sequential loss of cell cycle checkpoint control contributes to malignant transformation of murine embryonic fibroblasts induced by 20-methylcholanthrene, Journal of Cellular Physiology 224, no. 1 (2010): 49-58, https://doi.org/10.1002/jcp.22089. The above article, published online on 23 April 2010 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between journal Editor in Chief, Alexander Hutchison; and Wiley Periodicals LLC. Concerns were raised by a third party regarding image manipulation and duplication in the above article. An investigation by the publisher has confirmed the following image-related concerns: duplication and splicing of western blots in Figure 3B(i); rotation, resizing, and duplication of cellular sections in Figure 4; duplication of cellular sections between Figure 4 and Figure 6 C; duplication of western blots in Figure 5B; duplication of western blots in Figure 6 A; and duplication of cellular sections in Figure 6 C. Because the evidence of image manipulation is extensive and impacts several figures, the journal has determined that the results are fundamentally compromised, and so the journal must retract the article. The authors disagree with the retraction.

Biocompatible Hybrid Graphenic Thin Coatings on Flexible Substrates through Matrix-Assisted Pulsed Laser Evaporation (MAPLE).

This work reports the production of biocompatible thin layers for biomedical applications based on a graphene-like material (GL), a graphene-related material (GRM) obtained from carbon black. GL was combined in a hybrid fashion with polydopamine (pDA), a mussel-inspired water-resistant wet adhesive bonding obtained by the oxidative polymerization of dopamine (DA), and polyvinyl pyrrolidinone (PVP), a nontoxic synthetic polymer with intrinsic adhesion properties, to obtain a tighter adhesion of the thin layer to the substrate (silicone slices). Matrix-assisted pulsed laser evaporation (MAPLE) was used to coat PDMS slices with thin films of GL-pDA and GL-PVP directly from their frozen suspensions in water. The results indicate that the relevant chemical-physical characteristics of both thin films (evidenced by FTIR and AFM) were maintained after MAPLE deposition and that the films exhibit uniformity also at the nanometric level. After deposition, the GL-pDA and GL-PVP films underwent a biological survey toward murine fibroblasts (NIH3T3), human keratinocytes (HaCAT), and human cervical adenocarcinoma epithelial-like (HeLa) cells to assess the feasibility of this approach. Results indicate that both the GL-pDA and GL-PVP films did not perturb the biological parameters evaluated, including cytoskeleton alterations. Both hybrid films enhanced the effects of GL on cellular vitality across all cell lines. Specifically, the GL-pDA film exhibited a more stable effect over time (up to 72 h), whereas the GL-PVP film behaved similarly to the GL film in NIH3T3 and HeLa cell lines after long-term exposure. These promising results make the GL-pDA and GL-PVP films potential candidates for the manufacture of coated flexible devices for biomedical applications.

Localized in vivo gene editing of murine cancer-associated fibroblasts.

Discovering the role of fibroblasts residing in the tumor microenvironment (TME) requires controlled, localized perturbations because fibroblasts play critical roles in regulating immunity and tumor biology at multiple sites. Systemic perturbations can lead to unintended, confounding secondary effects, and methods to locally genetically engineer fibroblasts are lacking. To specifically investigate murine stromal cell perturbations restricted to the TME, we developed an adeno-associated virus (AAV)-based method to target any gene-of-interest in fibroblasts at high efficiency (>80%). As proof of concept, we generated single (sKO) and double gene KOs (dKO) of Osmr , Tgfbr2 , and Il1r1 in cancer-associated fibroblasts (CAFs) and investigated how their cell states and those of other cells of the TME subsequently change in mouse models of melanoma and pancreatic ductal adenocarcinoma (PDAC). Furthermore, we developed an in vivo knockin-knockout (KIKO) strategy to achieve long-term tracking of CAFs with target gene KO via knocked-in reporter gene expression. This validated in vivo gene editing toolbox is fast, affordable, and modular, and thus holds great potential for further exploration of gene function in stromal cells residing in tumors and beyond.

Cell-Type-Specific Effect of Innate Immune Signaling on Stress Granules

Stress granules (SGs) are cytoplasmic membraneless compartments that can form in stressed cells. There is an intricate relationship between SGs and innate immune signaling pathways. A previous study reported that the innate immune signaling mediated by Toll-like receptors (TLRs) can inhibit SGs induced by endoplasmic reticulum stress (ER stress) in bone-marrow-derived macrophages (BMDMs) and the chemotherapy drug oxaliplatin in B16 melanoma cells. We wanted to test if this observation can be generalized to other cell types. First, we recapitulated the results from the previous study showing TLR signaling-mediated inhibition of SGs in BMDMs induced by ER stress. However, SGs formed in response to ER stress were either not inhibited or only very weakly inhibited by TLR4 stimulation in human lung cancer-derived A549 cells, murine immortalized mouse lung fibroblasts (iMLFs) and primary murine mouse lung fibroblasts. This correlated with a weak induction of IKK complex kinase activity by TLR4 stimulation in these cells. SGs formed by sodium arsenite treatment also remained unaffected by TLR4 signaling. Our results indicate that the innate immune signaling-mediated inhibition of SGs is cell-type-dependent, thus opening a new avenue for mechanistic studies of the crosstalk between innate immune and stress signaling pathways.

Molecular epidemiology of enteroviruses from Guatemalan wastewater isolated from human lung fibroblasts.

The Global Specialized Polio Laboratory at CDC supports the Global Poliovirus Laboratory Network with environmental surveillance (ES) to detect the presence of vaccine strain polioviruses, vaccine-derived polioviruses, and wild polioviruses in high-risk countries. Environmental sampling provides valuable supplementary information, particularly in areas with gaps in surveillance of acute flaccid paralysis (AFP) mainly in children less than 15 years. In collaboration with Guatemala's National Health Laboratory (Laboratorio Nacional de Salud Guatemala), monthly sewage collections allowed screening enterovirus (EV) presence without incurring additional costs for sample collection, transport, or concentration. Murine recombinant fibroblast L-cells (L20B) and human rhabdomyosarcoma (RD) cells are used for the isolation of polioviruses following a standard detection algorithm. Though non-polio-Enteroviruses (NPEV) can be isolated, the algorithm is optimized for the detection of polioviruses. To explore if other EV's are present in sewage not found through standard methods, five additional cell lines were piloted in a small-scale experiment, and next-generation sequencing (NGS) was used for the identification of any EV types. Human lung fibroblast cells (HLF) were selected based on their ability to isolate EV-A genus. Sewage concentrates collected between 2020-2021 were isolated in HLF cells and any cytopathic effect positive isolates used for NGS. A large variety of EVs, including echoviruses 1, 3, 6, 7, 11, 13, 18, 19, 25, 29; coxsackievirus A13, B2, and B5, EV-C99, EVB, and polioviruses (Sabin 1 and 3) were identified through genomic typing in NGS. When the EV genotypes were compared by phylogenetic analysis, it showed many EV's were genomically like viruses previously isolated from ES collected in Haiti. Enterovirus occurrence did not follow a seasonality, but more diverse EV types were found in ES collection sites with lower populations. Using the additional cell line in the existing poliovirus ES algorithm may add value by providing data about EV circulation, without additional sample collection or processing. Next-generation sequencing closed gaps in knowledge providing molecular epidemiological information on multiple EV types and full genome sequences of EVs present in wastewater in Guatemala.

Sucrose and Glycerol Additives: A Way to Tune the Biological and Physicochemical Properties of Agarose Hydrogels?

AbstractSucrose and glycerol have gained attention as additives for hydrogels, owing to their capacity to exert considerable influence over the physicochemical, mechanical, and biological characteristics of these materials. Herein, these effects on agarose hydrogels (AHs) are explored. A series of AHs are synthesized using sucrose (30% and 300% w/v) and glycerol as additives. The storage modulus (10.0–13.7 kPa) and hydrophilicity of the hydrogels (contact angle < 50°) do not vary significantly with sucrose or glycerol addition. However, sucrose enhances the hydration capacity of the hydrogels by up to 170%, whereas glycerol reduces it. Interestingly, sucrose and glycerol individually do not have bacteriostatic effects against Staphylococcus epidermidis, but their combination significantly (p ≤ 0.001) inhibits the growth of both S. epidermidis and Pseudomonas aeruginosa by 63% and 29%, respectively, in comparison to native agarose. Cytotoxicity testing on NIH/3T3 murine fibroblasts reveals that sucrose increases cell viability up to 98%, while glycerol reduces it below 60%. Overall, these hydrogels hold promise for antibacterial biomedical applications as wound dressing materials and surface coatings for medical devices and can also be used to formulate bioinks for 3D bioprinting.

5,7,3′,4′-Tetramethoxyflavone suppresses TGF-β1-induced activation of murine fibroblasts in vitro and ameliorates bleomycin-induced pulmonary fibrosis in mice

Objective This study aimed to investigate the use of 5,7,3′,4′-tetramethoxyflavone (TMF) to treat pulmonary fibrosis (PF), a chronic and fatal lung disease. In vitro and in vivo models were used to examine the impact of TMF on PF. Methods NIH-3T3 (Mouse Embryonic Fibroblast) were exposed to transforming growth factor‑β1 (TGF-β1) and treated with or without TMF. Cell growth was assessed using the MTT method, and cell migration was evaluated with the scratch wound assay. Protein and messenger ribonucleic acid (mRNA) levels of extracellular matrix (ECM) genes were analyzed by western blotting and quantitative reverse transcription-polymerase chain reaction (RT–PCR), respectively. Downstream molecules affected by TGF-β1 were examined by western blotting. In vivo, mice with bleomycin-induced PF were treated with TMF, and lung tissues were analyzed with staining techniques. Results The in vitro results showed that TMF had no significant impact on cell growth or migration. However, it effectively inhibited myofibroblast activation and ECM production induced by TGF-β1 in NIH-3T3 cells. This inhibition was achieved by suppressing various signaling pathways, including Smad, mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/AKT (PI3K/AKT), and WNT/β-catenin. The in vivo experiments demonstrated the therapeutic potential of TMF in reducing PF induced by bleomycin in mice, and there was no significant liver or kidney toxicity observed. Conclusion These findings suggest that TMF has the potential to effectively inhibit myofibroblast activation and could be a promising treatment for PF. TMF achieves this inhibitory effect by targeting TGF-β1/Smad and non-Smad pathways.

Optimization of enzymatic production of anti-skin aging biopeptides from white sorghum [Sorghum bicolor (L) Moench] grain

Recently, kafirins from white sorghum [Sorghum bicolor (L) Moench] grain have shown promise as a source of biopeptides with anti-skin aging effects (anti-inflammatory, antioxidant, and inhibition of photoaging-associated enzymes). This study employed response surface methodology (RSM) to optimize the extraction and enzymatic hydrolysis of kafirins (KAF) for the production of peptides with anti-skin aging properties. The optimization of conditions (reaction time and enzyme/substrate ratio) for liquefaction with α-amylase and hydrolysis of KAF with alcalase was performed using 32 complete factorial designs. Subsequently, ultrafiltered peptide extracts were obtained with molecular weights of 1–3 kDa (KAF-UF3) and lower than 1 kDa (KAF-UF1), which mainly contain hydrophobic amino acids (proline, leucine, isoleucine, phenylalanine, and valine) and peptide fractions with molecular weights of 0.69, 1.14, and 1.87 kDa. Consequently, the peptide extracts protected immortalized human keratinocytes (HaCaT cells) from ultraviolet B radiation (UVB)-induced damage by preventing the decrease and/or restoring the activity of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px)]. Furthermore, KAF-UF3 and KAF-UF1 inhibited (20–29%) elastase and collagenase overactivity in UVB-exposed murine fibroblasts (3T3 cells). Thus, KAF-UF3 and KAF-UF1 exhibited behavior similar to that observed with glutathione (GSH), suggesting their potential as functional peptide ingredients in skincare products.

Polyfunctionalized organoselenides: New synthetic approach from selenium-containing cyanohydrins and anti-melanoma activity

A series of seleno-containing polyfunctionalized compounds was synthesized exploring cyanohydrin chemistry, including α-hydroxy esters, α-hydroxy acids, 1,2-diols, and 1,2-diacetates, with yields ranging from 26 up to 99 %. The cytotoxicity of all synthesized compounds was then evaluated using a non-tumor cell line (BALB/3T3 murine fibroblasts), and those deemed non-cytotoxic had their anti-melanoma activity evaluated using B16-F10 murine melanoma cells. These assays identified two compounds with selective cytotoxic activity against the tested melanoma cell line, showing a potential anti-melanoma application.

Reovirus infection induces transcriptome-wide unique A-to-I editing changes in the murine fibroblasts.

The conversion of Adenosine (A) to Inosine (I), by Adenosine Deaminases Acting on RNA or ADARs, is an essential post-transcriptional modification that contributes to proteome diversity and regulation in metazoans including humans. In addition to its transcriptome-regulating role, ADARs also play a major part in immune response to viral infection, where an interferon response activates interferon-stimulated genes, such as ADARp150, in turn dynamically regulating host-virus interactions. A previous report has shown that infection from reoviruses, despite strong activation of ADARp150, does not influence the editing of some of the major known editing targets, while likely editing others, suggesting a potentially nuanced editing pattern that may depend on different factors. However, the results were based on a handful of selected editing sites and did not cover the entire transcriptome. Thus, to determine whether and how reovirus infection specifically affects host ADAR editing patterns, we analyzed a publicly available deep-sequenced RNA-seq dataset, from murine fibroblasts infected with wild-type and mutant reovirus strains that allowed us to examine changes in editing patterns on a transcriptome-wide scale. To the best of our knowledge, this is the first transcriptome-wide report on host editing changes after reovirus infection. Our results demonstrate that reovirus infection induces unique nuanced editing changes in the host, including introducing sites uniquely edited in infected samples. Genes with edited sites are overrepresented in pathways related to immune regulation, cellular signaling, metabolism, and growth. Moreover, a shift in editing targets has also been observed, where the same genes are edited in infection and control conditions but at different sites, or where the editing rate is increased for some and decreased for other differential targets, supporting the hypothesis of dynamic and condition-specific editing by ADARs.

Tissue-engineered cellulose tubes for microvascular and lymphatic reconstruction: A translational and feasibility study

BackgroundLymphedema microsurgery is an emerging treatment modality, with dissimilar long-term outcomes. One of the main technical challenges in lymphatic microsurgery is the identification and availability of suitable donor vessels for anastomosis. Tissue engineering using biomaterials has shown promise in addressing vessel quality issues in other fields, but its application in microsurgery is still limited. MethodsDecellularized cellulose tubes were developed and bioengineered by decellularizing stems of Taraxacum-Ruderalia. The microscopic structure, mechanical properties, and remnant DNA content of the cellulose tubes were evaluated. Human and murine skin fibroblasts and dermal lymphatic endothelial cells were isolated and cultured for recellularization studies. Biocompatibility, proliferative capacity, and ex-vivo endothelialization of the cellulose tubes were assessed as potential interposition grafts. Finally, the engineered cellulose tubes were assessed as interposing xenografts for LVA in an ex-vivo swine limb model. ResultsThe decellularized cellulose tubes exhibited a suitable microscopic structure, mechanical properties, and low remnant DNA content. The tubes showed adequate biocompatibility, supported cell proliferation, and facilitated spontaneous ex-vivo endothelialization of lymphatic endothelial cells. In the swine limb model, LVA using the engineered cellulose tubes was successfully performed. ConclusionThis translational study presents the use of decellularized cellulose tubes as an adjunct for micro and supermicrosurgical reconstruction. The developed tubes demonstrated favorable structural, mechanical, and biocompatible properties, making them a potential candidate for improving long-term outcomes in lymphedema surgical treatment. The next translational step would be trialing the obtained tubes in a microsurgical in-vivo model.

A Nonclinical Safety Evaluation of Cold Atmospheric Plasma for Medical Applications: The Role of Genotoxicity and Mutagenicity Studies.

Atmospheric nonthermal plasma (ANTP) has rapidly evolved as an innovative tool in biomedicine with various applications, especially in treating skin diseases. In particular, the formation of reactive oxygen species (ROS) and nitrogen species (RNS), which are generated by ANTP, plays an important role in the biological signaling pathways of human cells. Unfortunately, excessive amounts of these reactive species significantly result in cellular damage and cell death induction. To ensure the safe application of ANTP, preclinical in vitro studies must be conducted before proceeding to in vivo or clinical trials involving humans. Our study aimed to investigate adverse effects on genetic substances in murine fibroblast cells exposed to ANTP. Cell viability and proliferation were markedly reduced after exposing the cells with plasma. Both extracellular and intracellular reactive species, especially RNS, were significantly increased upon plasma exposure in the culture medium and the cells. Notably, significant DNA damage in the cells was observed in the cells exposed to plasma. However, plasma was not classified as a mutagen in the Ames test. This suggested that plasma led to the generation of both extracellular and intracellular reactive species, particularly nitrogen species, which affect cell proliferation and are also known to induce genetic damage in fibroblast cells. These results highlight the genotoxic and mutagenic effects of ANTP, emphasizing the need for the cautious selection of plasma intensity in specific applications to avoid adverse side effects resulting from reactive species production.

Donkey Gelatin and Keratin Nanofibers Loaded with Antioxidant Agents for Wound Healing Dressings.

Acute and chronic wounds present a significant healthcare challenge, requiring innovative solutions for effective treatment. The exploitation of natural by-products with advanced cell regeneration potential and plant-based materials, which possess bioactive properties, is an innovative topic in wound management. This study investigates the potential of donkey gelatin and keratin for blending with natural bioactive extracts such as sumac, curcumin, and oak acorn to fabricate antioxidant and antimicrobial nanofibers with accelerated wound healing processes. The fabricated nanofibers possess good in vitro biocompatibility, except for the sumac-based donkey nanofibers, where cell viability significantly dropped to 56.25% (p < 0.05 compared to non-treated cells). The nanofiber dimensions showed structural similarities to human extracellular matrix components, providing an ideal microenvironment for tissue regeneration. The donkey nanofiber-based sumac and curcumin extracts presented a higher dissolution in the first 10 min (74% and 72%). Curcumin extract showed similar antimicrobial and antifungal performances to rivanol, while acorn and sumac extracts demonstrated similar values to each other. In vitro tests performed on murine fibroblast cells demonstrated high migration rates of 89% and 85% after 24 h in the case of acorn and curcumin nanofibers, respectively, underscoring the potential of these nanofibers as versatile platforms for advanced wound care applications.

ATP-elicited Cation Fluxes Promote Volume-regulated Anion Channel LRRC8/VRAC Transport cGAMP for Antitumor Immunity.

The cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway is instrumental to antitumor immunity, yet the underlying molecular and cellular mechanisms are complex and still unfolding. A new paradigm suggests that cancer cells' cGAS-synthesized cGAMP can be transferred to tumor-infiltrating immune cells, eliciting STING-dependent IFN-β response for antitumor immunity. Nevertheless, how the tumor microenvironment may shape this process remains unclear. In this study, we found that extracellular ATP, an immune regulatory molecule widely present in the tumor microenvironment, can potentiate cGAMP transfer, thereby boosting the STING signaling and IFN-β response in murine macrophages and fibroblasts. Notably, genetic ablation or chemical inhibition of murine volume-regulation anion channel LRRC8/volume-regulated anion channel (VRAC), a recently identified cGAMP transporter, abolished ATP-potentiated cGAMP transfer and STING-dependent IFN-β response, revealing a crucial role of LRRC8/VRAC in the cross-talk of extracellular ATP and cGAMP. Mechanistically, ATP activation of the P2X family receptors triggered Ca2+ influx and K+ efflux, promoting reactive oxygen species production. Moreover, ATP-evoked K+ efflux alleviated the phosphorylation of VRAC's obligate subunit LRRC8A/SWELL1 on S174. Mutagenesis studies indicated that the phosphorylation of S174 on LRRC8A could act as a checkpoint for VRAC in the steady state and a rheostat of ATP responsiveness. In an MC38-transplanted tumor model, systemically blocking CD39 and ENPP1, hydroxylases of extracellular ATP and cGAMP, respectively, elevated antitumor NK, NKT, and CD8+ T cell responses and restrained tumor growth in mice. Altogether, this study establishes a crucial role of ATP in facilitating LRRC8/VRAC transport cGAMP in the tumor microenvironment and provides new insight into harnessing cGAMP transfer for antitumor immunity.

Photocrosslinkable hydrogel of ibuprofen-chitosan methacrylate modulates inflammatory response.

Methacrylated biopolymers are unique and attractive in preparing photocrosslinkable hydrogels in biomedical applications. Here we report a novel chitosan (CS) derivative-based injectable hydrogel with anti-inflammatory capacity via methacrylation modification. First, ibuprofen (IBU) was conjugated to the backbone of CS by carbodiimide chemistry to obtain IBU-CS conjugate, which converts water-insoluble unmodified CS into water-soluble IBU-CS conjugate. The IBU-CS conjugate did not precipitate at the pH of 7, which was beneficial to subsequent chemical modification with methacrylic anhydride to prepare IBU-CS methacrylate (IBU-CS-MA) with significantly higher methacrylation substitution. Photocrosslinkable in situ gel formation of injectable IBU-CS-MA hydrogel was verified using lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) initiator under visible light. The IBU-CS-MA hydrogel showed good cytocompatibility as revealed by encapsulating and in vitro culturing murine fibroblasts within hydrogels. It promoted macrophage polarization toward M2 phenotype, as well as downregulated pro-inflammatory gene expression and upregulated anti-inflammatory gene expression of macrophages. The hydrogel also significantly reduced the reactive oxygen specifies (ROS) and nitrogen oxide (NO) produced by lipopolysaccharides (LPS)-stimulated macrophages. Upon subcutaneous implantation in a rat model, it significantly mitigated inflammatory responses as shown by significantly lower inflammatory cell density, less cell infiltration, and much thinner fibrous capsule compared with CS methacrylate (CS-MA) hydrogel. This study suggests that IBU-CS conjugate represents a feasible strategy for preparing CS-based methacrylate hydrogels for biomedical applications.

Mechanisms of mammalian orthoreovirus-induced cell death and NF-kB activation in ER+ breast cancer.

e13171 Background: Estrogen receptor positive (ER+) breast cancer is one of the most commonly diagnosed types of breast cancer in the United States. Although the five-year survival rate of ER+ breast cancer is 90% or higher, ER+ breast cancer can recur and metastasize 10-20+ years after initial diagnosis. Novel therapeutic strategies are needed to prevent and treat breast cancer recurrence and metastasis. A possible solution is mammalian orthoreovirus (MRV), a replication-competent natural oncolytic virus. The strain T3D (Reolysin) is in current clinical trials to treat metastatic breast cancer; however, the mechanisms of MRV-induced death are not well elucidated. Furthermore, other MRV strains have not been well studied for their oncolytic potential in ER+ breast cancer. Our studies aim to explore the different cell death pathways elicited by MRV. Prior studies have reported that MRV induces cell death in L929 cells (murine fibroblasts, permissive to MRV infection) by activating apoptosis, necroptosis, and inhibiting the NFκB pathway to enhance viral replication. Methods: Our goal is to compare the effects of MRV in L929 cells and ER+ breast cancer cell line MCF7 using chemical inhibitors of necroptosis, apoptosis, and the NFκB pathway. Results: Our results indicate that necroptosis inhibitors do not impact cell viability in response to MRV infection, but apoptosis inhibitors increase cell viability, suggesting that MRV-induces apoptotic cell death in ER+ breast cancer cells. Previous work has shown that the NFκB pathway may be activated or inhibited by MRV. Our cell viability studies in MCF7 cells with the IKK inhibitor BAY-11-7082 indicate that several MRV strains likely activate the NFκB pathway to potentially enhance viral replication. Conclusions: Future studies are aimed at further defining mechanisms of MRV-induced cell death in ER+ breast cancer. We anticipate future studies will lead to an improved MRV virotherapeutic that may be combined with clinically relevant treatments, such as CDK4/6 inhibitors and anti-estrogens, to prevent and treat therapy-resistant ER+ breast cancer.