3T3 Mouse Fibroblasts Research Articles

Two Small Molecule Drugs with Topical Applications, Diflunisal and Naphazoline, and Their Potentially Toxic Photodegradants: Analysis by Chemical and Biological Methods.

Because of their topical application in patients and meaningful UV/VIS absorptive properties, the degradation and potential toxicity under irradiation of diflunisal (DIF) and naphazoline (NAF) were studied. In addition, the impact of pH on their photostability was examined, showing the highest degradation of acidic DIF at pH 1 and 13 and the highest degradation of basic NAF at pH below 7. An LC-UV analysis and chemical tests showed the first-order kinetics for their degradation and generation of reactive oxygen species (ROS). A UPLC-HRMS/MS analysis allowed us to identify four degradants of DIF (from DD-1 to DD-4) and six degradants of NAF (from ND-1 to ND-6). When Toxtree software was used, a high class III of toxicity was observed for DD-2, DD-3, and DD-4, and for all the NAF degradants. Furthermore, the ND-2 product, i.e., 2-[(1-methylnaphthalen-2-yl)methyl]-4,5-dihydro-1H-imidazole, was shown to present medium mutagenic and high tumorigenic effects according to OSIRIS Property Explorer. In addition, two in vitro tests on BALB/c 3T3 mouse fibroblasts showed a phototoxic effect of DIF and NAF at the lowest concentrations tested, i.e., 5 µg/mL. Thus, our present results could be useful to design further phototoxicity studies for DIF and NAF to minimize the risk of phototoxicity due to their photodegradation.

Thermally Stabilised Poly(vinyl alcohol) Nanofibrous Materials Produced by Scalable Electrospinning: Applications in Tissue Engineering.

Electrospinning is a widely employed manufacturing platform for tissue engineering applications because it produces structures that closely mimic the extracellular matrix. Herein, we demonstrate the potential of poly(vinyl alcohol) (PVA) electrospun nanofibers as scaffolds for tissue engineering. Nanofibers were created by needleless direct current electrospinning from PVA with two different degrees of hydrolysis (DH), namely 98% and 99% and subsequently heat treated at 180 °C for up to 16 h to render them insoluble in aqueous environments without the use of toxic cross-linking agents. Despite the small differences in the PVA chemical structure, the changes in the material properties were substantial. The higher degree of hydrolysis resulted in non-woven supports with thinner fibres (285 ± 81 nm c.f. 399 ± 153 nm) that were mechanically stronger by 62% (±11%) and almost twice as more crystalline than those from 98% hydrolysed PVA. Although prolonged heat treatment (16 h) did not influence fibre morphology, it reduced the crystallinity and tensile strength for both sets of materials. All samples demonstrated a lack or very low degree of haemolysis (<5%), and there were no notable changes in their anticoagulant activity (≤3%). Thrombus formation, on the other hand, increased by 82% (±18%) for the 98% hydrolysed samples and by 71% (±10%) for the 99% hydrolysed samples, with heat treatment up to 16 h, as a direct consequence of the preservation of the fibrous morphology. 3T3 mouse fibroblasts showed the best proliferation on scaffolds that were thermally stabilised for 4 and 8 h. Overall these scaffolds show potential as 'greener' alternatives to other electrospun tissue engineering materials, especially in cases where they may be used as delivery vectors for heat tolerant additives.

The role of TGF-β in the electrotactic reaction of mouse 3T3 fibroblasts in vitro.

Endogenous electric fields (EFs) serve as a crucial signal to guide cell movement in processes such as wound healing, embryonic development, and cancer metastasis. However, the mechanism underlying cell electrotaxis remains poorly understood. A plausible hypothesis suggests that electrophoretic or electroosmotic forces may rearrange charged components of the cell membrane, including receptors for chemoattractants which induce asymmetric signaling and directional motility. This study aimed to explore the role of Transforming Growth Factor Beta (TGFβ) signaling in the electrotactic reaction of 3T3 fibroblasts. Our findings indicate that inhibiting canonical and several non-canonical signaling pathways originating from the activated TGF-β receptor does not hinder the directed migration of 3T3 cells to the cathode. Furthermore, suppression of TGF-β receptor expression does not eliminate the directional migration effect of 3T3 cells in the electric field. Additionally, there is no observed redistribution of the TGF-β receptor in the electric field. However, our studies affirm the significant involvement of Phosphoinositide 3-Kinase (PI3K) in electrotaxis, suggesting that in our model, its activation is likely associated with factors independent of TGFβ action.

Biocompatibility assessment of organic semiconductor pigments epindolidione and quinacridone

Organic colorants epindolidione (EPI) and quinacridone (QUI) are commercially available hydrogen-bonded semiconductor pigments. Despite their suitable properties for bioelectronic applications, their biocompatibility has not been thoroughly examined yet. In this study, thin EPI and QUI layers were applied on well plates by vacuum deposition technique followed by short and long-term in vitro biocompatibility study. In vitro testing represents a relatively fast and cheap approach, especially suitable for screening testing and prioritization of materials for further research. LIVE/DEAD assay, cell cycle analysis, adhesion and morphology of NIH 3T3 mouse fibroblasts have been investigated up to 7 days using flow cytometry and fluorescent optical microscopy. In summary, no significant differences were observed in viability, cell morphology, or attachment capabilities between control cells and cells grown on the EPI or QUI surfaces or cells cultivated in medium harvested from EPI- or QUI-coated wells. Our results suggest that both pigments are highly biocompatible. The absence of adverse biological effects of EPI and QUI together with their low cost and availability indicate their high application potential in next-generation bioelectronic devices.

Dysregulation of miR-146a is associated with exacerbated inflammation, oxidative and endoplasmic reticulum stress in the progression of diabetic foot ulcer.

Recent evidence has implicated the role of microRNA-146a (miR-146a) in regulating inflammatory responses. In the present study, we investigated the role of miRNA-146a in the progression of diabetic foot ulcer (DFU) in type 2 diabetes mellitus patients (T2DM) and studied its correlation with stress mediators such as Endoplasmic Reticulum (ER) and oxidative stress. Ninety subjects were enrolled and evenly distributed among three groups: Controls (n = 30), T2DM without complications (n = 30) and T2DM with foot ulcers (n = 30). Subsequently, each group was further subdivided based on the University of Texas classification. Peripheral blood was collected from all the study subjects, while tissue biopsies were taken only from DFU patients. Total RNA from both PBMCs and wound tissues were isolated using miRNA isolation kit and qPCR was performed to check the expression of miR-146a, ER stress and oxidative stress markers. Our findings revealed a significant decrease in miR-146a expression among T2DM patients with Grade 2 and Grade 3 DFUs compared with those with Grade 0 and Grade 1 DFUs. Notably, inflammatory genes regulated by miR-146a, including TRAF6, IRAK-1 and ADAM, were all upregulated in T2DM patients with Grade 2 and Grade 3 DFUs. Moreover, reduced miR-146a levels were correlated with increased markers of ER stress and oxidative stress in Grade 2 and Grade 3 DFU patients. Furthermore, our in vitro experiment using mouse 3T3 fibroblasts demonstrated a downregulation of miR-146a following induction of hyperglycaemia, ER stress and oxidative stress in these cells. These findings suggest a potential link between diminished miR-146a expression and heightened oxidative and ER stress in T2DM patients with more severe grades of DFUs. Our results imply that targeting miR-146a may hold therapeutic promise for managing disease progression in DFU patients, as it could help alleviate oxidative and ER stress associated with diabetic complications.

Preventing Staphylococci Surgical Site Infections with a Nitric Oxide-Releasing Poly(lactic acid-co-glycolic acid) Suture Material.

Of the 27 million surgeries performed in the United States each year, a reported 2.6% result in a surgical site infection (SSI), and Staphylococci species are commonly the culprit. Alternative therapies, such as nitric oxide (NO)-releasing biomaterials, are being developed to address this issue. NO is a potent antimicrobial agent with several modes of action, including oxidative and nitrosative damage, disruption of bacterial membranes, and dispersion of biofilms. For targeted antibacterial effects, NO is delivered by exogenous donor molecules, like S-nitroso-N-acetylpenicillamine (SNAP). Herein, the impregnation of SNAP into poly(lactic-co-glycolic acid) (PLGA) for SSI prevention is reported for the first time. The NO-releasing PLGA copolymer is fabricated and characterized by donor molecule loading, leaching, and the amount remaining after ethylene oxide sterilization. The swelling ratio, water uptake, static water contact angle, and tensile strength are also investigated. Furthermore, its cytocompatibility is tested against 3T3 mouse fibroblast cells, and its antimicrobial efficacy is assessed against multiple Staphylococci strains. Overall, the NO-releasing PLGA copolymer holds promise as a suture material for eradicating surgical site infections caused by Staphylococci strains. SNAP impregnation affords robust antibacterial properties while maintaining the cytocompatibility and mechanical integrity.

Sustainable bioactive food packaging based on electrospun zein-polycaprolactone nanofibers integrated with aster yomena extract loaded halloysite nanotubes

Compostable zein-polycaprolactone (PZ) electrospun nanofiber integrated with different concentrations of Aster yomena extract loaded halloysite nanotubes (A. yomena-HNT) as bioactive nanofibrous food packaging is reported. SEM micrographs reveal heterogeneous nanofibers. A. yomena extract used in the study showed weak antioxidant activity with AAI and TEAC values of 0.229 and 0.346. In vitro, release profile over 7 days of A. yomena indicates a controlled, sustained, and prolonged release. The prepared nanofibers were effective against both gram-positive and gram-negative bacteria. The prepared composite nanofibers were rendered biocompatible and nontoxic when subjected to WST-1 and LDH assay after incubating with NIH 3T3 mouse fibroblast cell line. PZ-15 nanofiber packaging showed the best postharvest quality preservation in Black mulberry fruits after 4 days of storage at 25 °C and 85 % Rh. Moreover, the in vitro decomposition test reveals that the fabricated nanofibers decompose in the soil and do not pose as a threat to the environment.

A Comprehensive Study on Lathyrus tuberosus L.: Insights into Phytochemical Composition, Antimicrobial Activity, Antioxidant Capacity, Cytotoxic, and Cell Migration Effects.

In this study, in vitro antioxidant, antimicrobial, cytotoxic, and cell migration effects of phenolic compounds of Lathyrus tuberosus leaves, known in the Transylvanian ethnomedicine, were investigated. Ultra-high-performance liquid chromatography-tandem mass spectrometry was employed for the analysis of the ethanolic and aqueous extracts. The antimicrobial properties were determined using a conventional microdilution technique. Total antioxidant capacity techniques were used using cell-free methods and cell-based investigations. Cytotoxic effects were conducted on 3T3 mouse fibroblasts and HaCaT human keratinocytes using a multiparametric method, assessing intracellular ATP, total nucleic acid, and protein levels. Cell migration was visualized by phase-contrast microscopy, employing conventional culture inserts to make cell-free areas. Together, 93 polyphenolic and monoterpenoid compounds were characterized, including flavonoid glycosides, lignans, hydroxycinnamic acid, and hydroxybenzoic acid derivatives, as well as iridoids and secoiridoids. The ethanolic extract showed high antioxidant capacity and strong antimicrobial activity against Bacillus subtilis (MIC80 value: 354.37 ± 4.58 µg/mL) and Streptococcus pyogenes (MIC80 value: 488.89 ± 4.75 µg/mL). The abundance of phenolic compounds and the results of biological tests indicate the potential for L. tuberosus to serve as reservoirs of bioactive compounds and to be used in the development of novel nutraceuticals.

The Study on Timolol and Its Potential Phototoxicity Using Chemical, In Silico and In Vitro Methods.

Timolol (TIM) is a non-selective ß-adrenergic receptor antagonist used orally for the treatment of hypertension and heart attacks, and topically for treating glaucoma; lately, it has also been used in some specific dermatological problems. In the present study, its photodegradation and potential risk of phototoxicity were examined using chemical, in silico and in vitro methods. The UV/VIS irradiated solutions of TIM at pH 1-13 were subjected to LC-UV and UPLC-HRMS/MS analyses showing pseudo first-order kinetics of degradation and several degradation products. The structures of these photodegradants were elucidated by fragmentation path analysis based on high resolution (HR) fragmentation mass spectra, and then used for toxicity evaluation using OSIRIS Property Explorer and Toxtree. Potential risk of phototoxicity was also studied using chemical tests for detecting ROS under UV/VIS irradiation and in vitro tests on BALB/c 3T3 mouse fibroblasts (MTT, NRU and Live/Dead tests). TIM was shown to be potentially phototoxic because of its UV/VIS absorptive properties and generation ROS during irradiation. As was observed in the MTT and NRU tests, the co-treatment of fibroblasts with TIM and UV/VIS light inhibited cell viability, especially when concentrations of the drug were higher than 50 µg/mL.

Functionally multifaceted alginate/curdlan/agarose-based bilayer fibro-porous dressings for addressing full-thickness diabetic wounds

Full-thickness diabetic wounds are chronic injuries characterized by bleeding, excessive exude, and prolonged inflammation. Single-layer dressings fail to address their disturbed pathophysiology. Therefore, bilayer dressings with structural and compositional differences in each layer have gained attention. We hypothesized that natural polymer (alginate, curdlan, and agarose) based bilayer dressings with inherent healing properties could effectively resolve these issues. Hence, bilayer dressings were fabricated by electrospinning curdlan/agarose/ polyvinyl alcohol blend (top layer) on an alginate/agarose/polyvinyl alcohol-based lyophilized porous (bottom) layer. Ciprofloxacin was incorporated in both layers as a potential antibacterial drug. The bilayer dressing exhibited high swelling (~1300 %), biocompatibility (>90 % with NIH 3T3 and L929 mouse fibroblasts), and hemocompatibility (hemolysis <5 %). In vitro, scratch assay revealed a faster wound closure (~ 95–100 %) than control. Inhibition zone assay revealed antibacterial activity against Staphylococcus aureus and Escherichia coli. Real-time (in vitro) gene expression experiments performed using human THP-1 macrophages exhibited a significant increase in anti-inflammatory cytokines (4.51 fold in IL-10) and a decrease in pro-inflammatory cytokines (1.42 fold in IL-6) in comparison to lipopolysaccharide. Thus, fabricated dressings with high swelling, hemostatic, immunomodulatory, and antibacterial characteristics can serve as potential multifunctional and sustainable templates for healing full-thickness diabetic wounds.

One-step synthesis of orange-red emissive carbon dots: photophysical insight into their excitation wavelength-independent and dependent luminescence.

A low-temperature method was developed to synthesize orange-red luminescence phosphor-doped carbon dots (CDs) without complicated purification procedures. These CDs showed excitation wavelength-independent narrow emission (photo-luminescence quantum yield, Φf ∼ 12 to 22%) with single exponential time-resolved decay in weakly polar/non-polar solvents, indicating the presence of one kind of chromophore. In contrast, the same CDs showed excitation wavelength-dependent broad emission (Φf ∼ 1 to 8%) with multi-exponential fluorescence decay in polar solvents. These CDs exhibited poor solubility in polar solvents, resulting in CD aggregates contributed by excitation wavelength-dependent weak luminescence. The CDs embedded in polymethyl methacrylate (PMMA) polymer film displayed bright orange-red fluorescence under UV 365 nm illumination, indicating their potential application in solid-state luminescence. Further, an analytical method was developed for the naked-eye detection of trifluoracetic acid (red emission) and triethylamine (green emission) under UV 365 nm illumination with reversible two switch-mode luminescence. Additionally, this efficient orange-red luminescence of CDs was utilized for possible bioimaging applications with negligible cytotoxicity in 3T3 mouse fibroblast cells.

6‐Aryl‐Substituted Harmanium Derivatives as Light‐Up Probes for Fluorimetric DNA Detection and Staining of Eukaryotic Cells

AbstractNine 6‐aryl‐substituted harmanium derivatives were synthesized by Suzuki‐Miyaura coupling of 6‐bromoharmane with arylboronic acids and subsequent N2‐benzylation. Depending on the substitution pattern of the aryl group the emission maxima cover a range between λfl=445 and 529 nm with quantum yields of Φfl=&lt;0.01–0.92. The interactions with duplex DNA were investigated by photometric and fluorimetric titrations as well as CD and LD spectroscopy. These ligands bind to DNA by intercalation with binding constants of Kb=1–3×104 M−1. It was demonstrated by fluorescence microscopy that the 2‐benzyl‐6‐(3‐methoxyphenyl)harmanium derivative operates as a fluorescent light‐up probe in the complex biological environment of eukaryotic cells, namely in NIH 3T3 mouse fibroblasts, as it stains the perinuclear cytoplasm and distinct substructures of the nucleus preferentially as compared with the rest of the nucleoplasm.

The dynamics of the electrotactic reaction of mouse 3T3 fibroblasts

The molecular mechanisms behind electrotaxis remain largely unknown, with no identified primary direct current electric field (dcEF) sensor. Two leading hypotheses propose mechanisms involving the redistribution of charged components in the cell membrane (driven by electrophoresis or electroosmosis) and the asymmetric activation of ion channels. To investigate these mechanisms, we studied the dynamics of electrotactic behaviour of mouse 3T3 fibroblasts.We observed that 3T3 fibroblasts exhibit cathodal migration within just 1 min when exposed to physiological dcEF. This rapid response suggests the involvement of ion channels in the cell membrane. Our large-scale screening method identified several ion channel genes as potential key players, including the inwardly rectifying potassium channel Kir4.2. Blocking the Kir channel family with Ba2+ or silencing the Kcnj15 gene, encoding Kir4.2, significantly reduced the directional migration of 3T3 cells. Additionally, the levels of the intracellular regulators of Kir channels, spermine (SPM) and spermidine (SPD), had a significant impact on cell directionality. Interestingly, inhibiting Kir4.2 resulted in the temporary cessation of electrotaxis for approximately 1–2 h before its return. This observation suggests a two-phase mechanism for the electrotaxis of mouse 3T3 fibroblasts, where ion channel activation triggers the initial rapid response to dcEF, and the subsequent redistribution of membrane receptors sustains long-term directional movement.In summary, our study unveils the involvement of Kir channels and proposes a biphasic mechanism to explain the electrotactic behaviour of mouse 3T3 fibroblasts, shedding light on the molecular underpinnings of electrotaxis.

Fluorimetric Cell Analysis with 9-Aryl-Substituted Berberine Derivatives as DNA-Targeting Fluorescent Probes.

DNA-sensitive fluorescent light-up probes based on berberine are presented. This biogenic fluorophore was chosen as central unit to use its potential biocompatibility and its DNA-binding properties. To provide predictable fluorescence quenching in aqueous solution and a fluorescence light-up effect upon DNA binding, aryl substituents were attached at the 9-position by Suzuki-Miyaura coupling reactions. The 9-arylberberine derivatives have a very low fluorescence quantum yield (Φfl =<0.02), which is caused by the radiationless deactivation of the excited state by torsional relaxation about the biaryl axis. In addition, these berberine derivatives intercalate into DNA with high affinity (Kb =2.0-22×104 M-1 ). Except for the nitrophenyl- and hydroxyphenyl-substituted derivatives, all tested compounds exhibited a pronounced fluorescence light-up effect upon association with DNA, because the deactivation of the excited-state by torsional relaxation is suppressed in the DNA binding site. Most notably, it was shown exemplarily with the 9-(4-methoxyphenyl)- and the 9-(6-methoxynaphthyl)-substituted derivatives that these properties are suited for fluorimetric cell analysis. In particular, these probes generated distinct staining patterns in eukaryotic cells (NIH 3T3 mouse fibroblasts), which enabled the identification of nuclear substructures, most likely heterochromatin or nucleoli, respectively.

Biocompatibility of bulk-fill resins in vitro.

This study aims to evaluate the cytotoxicity and genotoxicity of three different extracts obtained from Filtek™ One Bulk Fill, Tetric Evoceram® Bulk Fill and Coltene Fill-Up! resins. The cytotoxicity was determined on 3T3 fibroblast cells using the MTT and crystal violet assays. The genotoxicity was determined using a cytokinesis-block micronucleus assay. The cytotoxicity of the resin extracts on 3T3 mouse fibroblasts was found to be dose-dependent with both the MTT and crystal violet assays. Extracts concentrated above 1% were cytotoxic according to the MTT assay. The Filtek™ One Bulk Fill, Tetric Evoceram® Bulk Fill, and Coltene Fill-Up! resins reached the LD50 at concentrations of 60%, 50%, and 20%, respectively, and showed genotoxicity rates that were 2-5 times, 3-8 times, and 4-15 times higher than the negative control, respectively. Coltene Fill-Up! resin extracts were the most cytotoxic and genotoxic, followed by Tetric Evoceram® Bulk Fill and Filtek™ One Bulk Fill. The analyzed bulk-fill resins showed differences in in vitro biocompatibility, and the Filtek™ One Bulk Fill was found to be the safest for clinical use.

Application of Crosslinking of Thiolated Hyaluronic Acid Nanogel for Targeted Therapy and Drug Delivery in Melanoma

Melanoma is extremely aggressive and its prevalence is growing every year. Gel was generated by altering thiolated hyaluronic acid on surface-functionalized Pluronic F127-TPGS mixed micelles to create a nanogel with the ability to selectively target melanoma. Cell uptake experiment was used to quantitatively and qualitatively assess the uptake of the nanogel by B16F10 melanoma cells; cytotoxicity experiment was used to investigate the toxicity of the carrier material to cells. Microscopic analysis of the produced nanogel revealed an average particle size of 30 nm, with no discernible cytotoxic effect on both mouse 3T3 fibroblasts and mouse melanoma B16F10 cells. Higher HCAM receptor expression in B16F10 cells allowed for more efficient absorption than in 3T3 cells.

Design, synthesis, in vitro urease inhibitory potentials and in silico molecular docking study of benzimidazole bearing thiosemicarbazides/sulfonamide Analogues

The nickel-containing urease enzyme is responsible for the pathogenesis of hepatic coma, hepatic encephalopathy, urolithiasis, gastric and peptic ulcer. These enzymes also have a negative effect on the efficacy of soil nitrogen to produce crops. The urease enzyme inhibitors may be thought as a strategy for reducing the negative effects of ureolytic bacteria. The present study involves a novel approach to the synthesis benzimidazole thiosemicarbazides and sulphonamide derivatives as potent urease inhibitor. All the analogues exhibited good inhibition potential. Among the thiosemicarbazides series, the most potent were analogs 1 g and 1 h having an IC50 = 2.40 ± 0.10 and 3.10 ± 0.10 µM respectively. Among the sulphonamide series, the most potent analogs were 2f and 2j having an IC50 = 3.90 ± 0.10 and 1.40 ± 0.001 µM respectively. Structure activity relationship study shows that among the two series, the most potent analogs were those having electron-withdrawing groups. Molecular docking study was carried out to check the interactions between the synthesized compounds and the urease enzyme's active sites. Furthermore, to evaluate the stability of the most active compound in complex with the urease enzyme a total of 200 ns MD simulation was carried out. The MD simulation study revealed that the compound formed a more stable complex with the urease enzyme and remained stable throughout the 200 ns MD simulation. All Compounds were verified for cytotoxicity against 3T3 mouse fibroblast cell line and detected nontoxic.

Enhanced antibacterial efficacy against antibiotic-resistant bacteria via nitric oxide-releasing ampicillin polymer substrates

The emergence of antibiotic-resistant bacteria poses a pressing threat to global health and is a leading cause of healthcare-related morbidity and mortality. Herein, we report the fabrication of medical-grade polymers incorporated with a dual-action S-nitroso-N-acetylpenicillamine-functionalized ampicillin (SNAPicillin) conjugated molecule through a solvent evaporation process. The resulting SNAPicillin-incorporated polymer materials act as broad-spectrum antibacterial surfaces that improve the administration efficacy of conventional antibiotics through the targeted release of both nitric oxide and ampicillin. The polymer surfaces were characterized by scanning electron microscopy and static contact angle measurements. The nitric oxide (NO) release profile and diffusion of SNAPicillin from polymers were quantified using a chemiluminescence-based nitric oxide analyzer (NOA) and ultraviolet–visible (UV–vis) spectroscopy. As a result, the films had up to 2.96 × 10−7 mol cm−2 of total NO released within 24 hr. In addition, >79 % of the SNAPicillin reservoir was preserved in the polymers after 24 hr of incubation in the physiological environment, indicating their longer-term NO release ability and therapeutic window for antibacterial effects. The SNAPicillin-incorporated polymers reduced the viability of adhered bacteria in culture, with >95 % reduction found against clinically relevant strains of Staphylococcus aureus (S. aureus). Furthermore, SNAPicillin-modified surfaces did not elicit a cytotoxic effect toward 3T3 mouse fibroblast cells, supporting the material's biocompatibility in vitro. These results indicate that the complementary effects of NO-release and ampicillin in SNAPicillin-eluting polymers can enhance the properties of commonly infected medical device surfaces for antibacterial purposes.

Gamma Secretase Inhibition Sensitizes Pancreatic Adenocarcinoma Tumors to RT In Vivo

Pancreatic adenocarcinoma (PDAC) is an extremely aggressive cancer that lacks curative treatment options. Almost half of patients present with unresectable disease limiting treatment to non-curative options. Patients treated with neoadjuvant radiation therapy (RT) exhibit increases in fibrosis and epithelial-to-mesenchymal transition (EMT). ADAM10, an extracellular sheddase, can stimulate stromal fibrosis, EMT, and radioresistance. ADAM10 also mediates EMT through Notch signaling by cleaving its extracellular domain. Further cleavage by gamma secretase produces the Notch intracellular domain (NICD), which translocates to the nucleus and activates downstream transcriptional targets. Here, we explore whether inhibition of Notch cleavage by gamma secretase radiosensitizes PDAC tumors. Bilateral flank subcutaneous PDAC isografts were produced in 40 mice using PK5L1940 KPC cells. Intraperitoneal injections of the gamma secretase inhibitor, DAPT (5 mg/kg), were delivered daily for 7 days, starting 3 days prior to RT. A single dose of 20 Gy was administered to each flank tumor, and volumes were measured twice weekly. Colony formation assays of KPC cells were performed after RT, in the presence or absence of DAPT. Since stromal fibrosis can mediate radio-resistance in the tumor microenvironment (TME), the effect of tumor cells on Notch pathway activation in mouse fibroblasts (3T3 cells) was investigated using a luciferase reporter assay. Thus, 3T3 cells transfected with a Notch pathway luciferase reporter were incubated with PDAC cells for 48 h, followed by measurement of luciferase activity. In vivo, the combination of DAPT and RT significantly delayed tumor growth, and some tumors were completely eradicated. Mean tumor size for the combination at 21 days was 21 mm3 (range = 0-53, p = 0.005), while tumor size was 577 mm3 (range = 217-955, p = 0.69) for DAPT alone, 435 mm3 for RT alone (range = 51-932, p = 0.79), and 367 mm3 for untreated vehicle (range = 97-1144). Surprisingly, DAPT did not reduce clonogenic survival in vitro. Both ADAM10 knockout and DAPT decreased NICD cleavage and transcription of the downstream target Hes1 in vivo and in vitro. Co-culture with PDAC cells increased Notch luciferase reporter activity in fibroblasts. This effect was not mimicked by PDAC-conditioned media, suggesting a requirement for intercellular contact. Notch pathway inhibition sensitizes PDAC tumors to RT in vivo, but not in vitro, suggesting involvement of the TME. Indeed, co-culture with PDAC cells stimulates notch signaling in fibroblasts, suggesting non-cell autonomous mechanisms mediating fibrosis in the TME driving radioresistance. Future studies will determine if ADAM10 inhibition targeting PDAC cells and/or gamma secretase inhibition targeting the TME enhances radiation sensitivity in vivo by blocking fibroblast Notch signaling.

Oct4 and Hypoxia Dual-Regulated Oncolytic Adenovirus Armed with shRNA-Targeting Dendritic Cell Immunoreceptor Exerts Potent Antitumor Activity against Bladder Cancer.

Immunotherapy has emerged as a promising modality for cancer treatment. Dendritic cell immunoreceptor (DCIR), a C-type lectin receptor, is expressed mainly by dendritic cells (DCs) and mediates inhibitory intracellular signaling. Inhibition of DCIR activation may enhance antitumor activity. DCIR is encoded by CLEC4A in humans and by Clec4a2 in mice. Gene gun-mediated delivery of short hairpin RNA (shRNA) targeting Clec4a2 into mice bearing bladder tumors reduces DCIR expression in DCs, inhibiting tumor growth and inducing CD8+ T cell immune responses. Various oncolytic adenoviruses have been developed in clinical trials. Previously, we have developed Ad.LCY, an oncolytic adenovirus regulated by Oct4 and hypoxia, and demonstrated its antitumor efficacy. Here, we generated a Clec4a2 shRNA-expressing oncolytic adenovirus derived from Ad.LCY, designated Ad.shDCIR, aimed at inducing more robust antitumor immune responses. Our results show that treatment with Ad.shDCIR reduced Clec4a expression in DCs in cell culture. Furthermore, Ad.shDCIR exerted cytolytic effects solely on MBT-2 bladder cancer cells but not on normal NIH 3T3 mouse fibroblasts, confirming the tumor selectivity of Ad.shDCIR. Compared to Ad.LCY, Ad.shDCIR induced higher cytotoxic T lymphocyte (CTL) activity in MBT-2 tumor-bearing immunocompetent mice. In addition, Ad.shDCIR and Ad.LCY exhibited similar antitumor effects on inhibiting tumor growth. Notably, Ad.shDCIR was superior to Ad.LCY in prolonging the survival of tumor-bearing mice. In conclusion, Ad.shDCIR may be further explored as a combination therapy of virotherapy and immunotherapy for bladder cancer and likely other types of cancer.