N,N-dimethylacrylamide, DMA) and temperature-sensitive  units. By scavengingReactive Oxygen Species (ROS), these polymers are anti-inflammatory;through temperature responsiveness, they can macroscopically aggregate,which may allow them to form depots upon injection. The localizationof the anti-inflammatory action is an example of synergy. An extensiveevaluation of toxicity and anti-inflammatory effects on invitro models, including BV2 microglia, C8D30 astrocytes andprimary neurons, shows a link between capacity of aggregation anddetrimental effects on viability which, albeit mild, can hinder theanti-inflammatory potential (antagonist action). Although limitedin breadth , this study suggests that single-responsivecontrols should be used to allow for a precise assessment of the (synergicor antagonist) potential of double-responsive systems.This study is aboutmultiple responsiveness in biomedical materials.This typically implies “orthogonality”  or synergy , but an antagonist effect between responsesmay also occur. Here, we describe a family of very well-defined amphiphilicand micelle-forming block copolymers, which show both oxidative andtemperature responses. They are produced via successive anionic ring-openingpolymerization of episulfides and RAFT polymerization of dialkylacrylamidesand differ only in the ratio between inert ( For example, temperature-responsive systems composed of poly(ethyleneglycol) (PEG)–poly(lactic-co-glycolic acid)(PLGA)9 and polymers of oligo- and di(ethyleneglycol) methyl ether methacrylates10 wereshown to be efficacious systems for in situ, body-temperature-activatedformation of drug-loaded matrices in the treatment of spinal cordinjury.Inflammation is a natural response ofthe immune system to protectthe body from damage potentially caused by pathogens or disruptedcellular processes. When controlled, it aims to restore homeostasis.When chronic, it is typically associated with a vast array of detrimentaleffects and an increased levels of oxidants;in situ depot formation in the context of polymers withinherent anti-inflammatory activity through Reactive Oxygen Species(ROS) scavenging.11 Our group has specificallystudied organic polysulfides, in particular poly(propylene sulfide)(PPS), but also polysulfoxides;12 we referthe reader to a recent review for an updated account on these andother sulfur-based ROS scavenging polymers.13 The oxidation of hydrophobic thioethers (sulfides) by ROS, producingpolar sulfoxides or sulfones, swells or solubilizes these nanomaterials,thereby allowing for an inflammatory-driven release of any encapsulateddrug, which can synergize or add to the direct anti-inflammatory effectsof ROS removal.14 Here, using invitro models of CNS inflammation, we aimed to ascertain anysynergistic or antagonistic outcomes of oxidant scavenging and temperature-inducedaggregation.Herein, we explore the potential of temperature-dependent 15 to self-assembled amphiphilicaggregates14) in a variety of multiblockcombinations with PEG. However, in the last 15 years, it has becomeincreasingly evident that anti-PEG antibodies naturally occur, arerather widespread, and lead to the accelerated blood clearance (ABC)of PEGylated formulations.17 In this study, as aPEG replacement, we have focused on poly, in particular poly (PDMA) and poly (PDEA). For example, whendecorating the surface of liposomes, PDMA has been shown not to induceABC effect or IgM production upon repeated administration in rats18 or poly(N-vinylpyrrolidone)).Such double temperature- and oxidation-responsivemacromoleculestypically have amphiphilic structures. Here, we have also focusedon the biocompatibility of their hydrophilic components. Poly(ethyleneglycol) (PEG) is a gold standard in this area, due to its biocompatibilityand “stealth” character; indeed, in previous studieswe have produced PPS in a variety of morphologies  (PNIPAm) diblocks.23 The latter composition combined the hydrophilicity and biocompatibilityof PDMA with the inverse temperature gelation of PNIPAm, which mayallow in situ gelation after injection and therebythe spatial resolution of the ROS scavenging activity of the system.For this very purpose, oxidation/temperature double responsive systemshave been studied in recent years chiefly using PNIPAm24 or related systems.25 However, the literaturein the field is scarce in providing comprehensive studies, lackingfor example negative controls that depict no thermal gelation forcomparison of such systems.Indeed, PDMAhas been widely employed as an alternative to PEG.26 functionally similar to PNIPAm.27 It exhibits a lower critical solution temperature (LCST)in the region of 32–34 °C, slightly decreasing with molecularweights >104 Da,28 and broadlyindependent of the macromolecular architecture . In DEA/DMA random copolymers, DMA contentincreases the LCST.31 If this role is maintainedalso in PPS-containing structures, this parameter may provide a finecontrol over the range of thermal response.PDEA, on the one hand, is structurallyanalogous to PDMA  but,on the other hand, is a thermally sensitive macromolecule,in situ gelation) may present any adverseor synergic biological effects in the perspective of a CNS anti-inflammatorytherapy. Such biomedical relevance was evaluated in terms of cellviability and inflammation biomarkers in 2D in vitro models with four different cell lines, namely macrophages, fibroblasts,astrocytes, and microglia, as well as with primary neurons, to achievea comprehensive overview of this family of doubly responsive materials.Herein, we presenta comprehensive structure–activity relationship,linking ROS scavenging activity and thermal gelation with DEA/DMAratio for a family of precisely designed polymeric PPS-based amphiphiles.For their synthesis, we have sequentially combined two “living”polymerization mechanisms: the anionic ring-opening polymerization(ROP) of episulfides yields macromolecular chain transfer agents (macroRAFTagents) used in the reversible addition, fragmentation, and termination(RAFT) of the acrylamidic monomers 1. By varOf note, to identify the best performing PPS-MacroRAFT agent (PMA)structure, polymers with different substituents on the trithiocarbonategroup were produced , in additionto a negative control without trithiocarbonate (BP-NEG). The overallaim was to produce well-defined macromolecules differing only in theDEA/DMA ratio and therefore in their potential for large-scale aggregationin water.N,N-dimethylformamide (DMF), tributylphosphine (TBP), and methanol werepurchased from Thermo Fisher Scientific . Sodiumsulfate anhydrous (Na2SO4), acetic acid, basicalumina, molecular sieves 4 Å, and 30 wt % hydrogen peroxidesolution (H2O2) were purchased from Merck . Nile Red was purchased from FluoroChem . Allother chemicals and solvents were purchased from Sigma-Aldrich . The inhibitor (monomethyl ether hydroquinone) was removed from N,N-dimethylacrylamide (DMA) and N,N-diethylacrylamide (DEA) by stirringwith inhibitor removers (for removing hydroquinone and monomethylether hydroquinone) for an hour at room temperature followed by columnfiltration through basic alumina. The monomers DEA and DMA were storedat −20 °C prior to reactions. DMF was dried overnighton molecular sieves 4 Å. 2,2′-azobis(2-methylpropionitrile)(AIBN) was recrystallized from methanol. The rest of the chemicalswere used without further purification.All chemicalswere used as received from suppliers unless otherwise stated. Dichloromethane(DCM), tetrahydrofuran (THF), Escherichia coli 02B:B6, Hank’sbalanced salt solution (HBSS), neurobasal medium, GlutaMAX, PierceBCA Protein Assay Kit, CyQUANT MTT cell viability assay, and TNF-αMouse Uncoated ELISA Kit were purchased from Thermo Fisher Scientific; RAW264.7 macrophages, BV2+ microglia, and C8D30astrocytes were purchased from ATCC. Dulbecco’s modified Eagle’smedium (DMEM), DMEM-high glucose (no phenol red), fetal bovine serum(FBS), l-glutamine 200 mM (×100), penicillin–streptomycin,and poly(l-lysine) (PLL) were purchased from Invitrogen . Cytotoxicity Detection Kit (LDH) was purchased from Roche ). The MTS proliferation assay (CellTiter96 Aqueous One Solution Cell Proliferation Assay) was purchased fromPromega . C57BL/6J mice were purchased from CharlesRiver Laboratories Italia s.r.l. .Human dermal fibroblasts, lipopolysaccharides(LPS) from 1H NMR spectrawere recorded on 15 mg/mL solutions in deuterated chloroform usinga 400 MHz Bruker spectrometer . 13C NMR spectra were recorded on 4-mg/mL solutions in deuterated chloroformusing a 500 MHz Bruker spectrometer .Spectra were recorded in ATR mode(Golden Gate) on a Tensor 27 Bruker spectrometer  equipped with a 3000 Series TM High Stability Temperature Controllerwith RS232 Control .M̅n and M̅w were directlycalculated from the peak list of the mass spectra by the Bruker MSsoftware PolyTools 2.0. The optimization of the analytical conditionsis described in Supporting Information, section 1.5SI and Figures S5 to S13.MALDI-ToF mass spectra were recordedon a Bruker UltrafleXtreme apparatus, operating in positive ionizationmode at 20 kV and 80% laser output. PPS macroRAFT agents were dissolvedin THF at a concentration of 10 mg/mL. The THF solutions containedalso 4′-hydroxyazobenzene-2-carboxylic acid or dithranol ata 0.1 M concentration in the presence of 0.1 M of NaTFA or no salt.Appropriate volumes of sample and matrix solutions were mixed to obtainratios of 1:1 or 1:2 (sample/matrix v/v). Appropriate volumes of NaTFAor THF  were added to the sample and matrixmixture to obtain a 1:10 ratio . An aliquotof 2 μL of each sample/matrix mixture was spotted onto a BrukerBigAnchor steel and slowly dried to allow matrix crystallization atambient conditions. Average molecular weights The analysis wasperformed using triple detection conditions with either a GPC50  or a Malvern OmniSec System . Molecular weight and molecular weight distributionof PPS polymers were determined using a PLgel 5 μm MIXED-D columnin series with a PLgel 3 μm MIXED-E operating online at 30 °C.THF was used as an eluent at a flow rate of 1.0 mL/min. Two linearpolystyrene standards  for calibration (narrow) and forverification (broad) were used for the analysis of the polymers. Blockcopolymers of PPS with polyacrylamides were analyzed using a D3000column operating online at 50 °C with HPLC grade DMF containing0.1% LiBr as the mobile phase at a flow rate of 0.8 mL/min. Calibrationwas performed using a poly(methyl methacrylate) standard.G′ (storage modulus)= G′′ (loss modulus) in experimentswhere the temperature was increased 2 °C/min in the interval20–60 °C, using a HAAKE RheoStress 6000  rheometer equipped with a Peltier temperaturecontrol unit. A cone–plate geometry with a diameter of 60 mmand a cone angle of 1° was used with a gap of 52 μm. Allmeasurements were performed in the linear viscoelastic region .Dispersions (250 mg/mL) were preparedby mixing appropriate quantities of the polymers and deionized waterat room temperature. The gel points of these dispersions were determinedas the temperature where Analysiswas performedon micellar dispersions at a concentration of 1 mg/mL in deionizedwater using Zetasizer Nano ZS Instrument  at an angle of 173° and a temperature of 25 °C.Micellar dispersions(0.1 mg/mL) of the polymers in deionized water were dropcast ontoultrathin carbon/Formvar coated copper 150 mesh grids and stainedwith 1% uranyl acetate in water for 60 s before being analyzed witha JEOL JEM-1011 TEM, equipped with a W filament operating at 100 kV.–5 M solution of Nile Red in THF in eachwell of a 96-well plate. The plate was protected from light, and THFwas allowed to evaporate overnight. The Nile Red fluorescence wasrecorded on a Synergy2 Biotek plate reader with Gen5 software at 37°C, using an excitation filter at 540 ± 25 nm and an emissionfilter at 620 ± 40 nm. The emission intensityat high and low concentration was fitted with linear equations, andtheir intersection was defined as the CAC (n = 3).Block copolymer dispersions, 200 μL in deionized water (concentrationsranging from 0.0001 to 10 mg/mL), were mixed with 10 μL of a3 × 10n = 3).Block copolymer dispersionsin deionized water (200 μL) at concentrations of 1, 10, 50,100, and 200 mg/mL in a 96-well plate were gradually heated from 24to 45 °C. The optical densities at 500 nm were recorded on aSynergy2 Biotek plate reader with Gen5 software at every 1 °Cincrease in temperature with 2 min equilibration at each temperaturebefore measurement (n = 3).Nile Red-loaded polymer dispersionsin deionized water were prepared and introduced in wells of a 96-wellplate and mixed with hydrogen peroxide, obtaining a final volume of200 μL, a thioether concentration of 5 mM (corresponding toa 0.4 mg/mL polymer concentration), and a hydrogen peroxide concentrationof 40 mg/mL . Controls without hydrogen peroxide wereused to consider the possible decrease of Nile Red emission over timeand were used to estimate the fraction of unbleached dye at all timepoints. The normalized fluorescence fraction was calculated as the[emission intensity at a given point] ÷ [emission intensity attime = 0 × the fraction of unbleached dye]  were prepared in smallvials. The samples were heated in an incubator from 25 to 40 °Cwith equilibration time of 10 min.in vacuo, and the product was dissolvedin 30 mL DCM and extracted 3 times with 6 mL brine, dried over Na2SO4, filtered, and concentrated in vacuo. Each concentrate was then extracted 3 times with 5 mL of methanol.The resulting oil was dried in a Genevac EZ2 Elite centrifugal evaporatorovernight at 40 °C. The final products were analyzed by 1H NMR, 13C NMR, FT-IR, GPC, and MALDI-ToF. Massrecovery (wt %) was calculated as the mass of polymer recovered dividedby the theoretical mass of the polymer as determined using the compositionmeasured by 1H NMR  × 100.All experiments were performedin a 12-position Radley’s carousel parallel reactor, purgingit with argon for 5 min prior to the reactions. THF was degassed for45 min with argon prior to the reactions. After each reaction, volatileswere removed DOTAc  , TBP ,and 1.2 mL  of a 0.5 M sodiummethoxide solution in methanol were added in 10 mL of THF. The mixturewas stirred for 15 min, then 850 mg of PS was introduced in the mixture, and the reagents were allowedto react for 45 min at room temperature. 0.14 mL  of carbon disulfide was introduced and allowed toreact for a further 15 min. Benzyl bromide (BB)  was added and reacted overnight at room temperature;722 mg (yield 66 wt %) of a yellow oily material was obtained.1H NMR (CDCl3): δ = 1.35–1.45, 2.55–2.70 , 2.71–2.78 , 2.85–3.05 , 3.61–3.68 ,4.58–4.65 –S–CH2–Ph), 7.30–7.38 ppm .13C NMR (CDCl3): δ = 20.8 (CH3 in PPS chain), 32.4 (−S–CH2–CH2–O–CH2–CH2–O–CH2–CH2–S−), 38.4 (CH2 in PPS chain), 41.2 (CH in PPS chain), 46.6 (CH2 in benzyl),70.4 (−S–CH2–CH2–O–CH2–CH2–O–CH2–CH2–S−), 71.2 (−S–CH2–CH2–O–CH2–CH2–O–CH2–CH2–S−), 127.8 (phenyl carbonin para position), 128.7 (phenyl carbons in ortho position), 129.3 (phenyl carbons in meta position), 135 (phenyl carbon in ipso position),222.6 ppm (−C(=S)–S–CH2–Ph).as CH3), 2919 (νas CH2), 2865 (νs CH3 and νs CH2), 1370(νas C–O–C), 820 (νas C=S), 734 cm–1 (νas C–S).FT-IR (ATR): ν̅ = 2958 : M̅n = 3700 g/mol, Đ = 1.01.MALDI-ToF (HABA matrix with NaTFAat 1:2 polymer-to-matrix ratioin THF): DOTAc , TBP ,and 1.2 mL  of a 0.5 M sodiummethoxide solution in methanol were added in 5 mL of THF. The mixturewas stirred for 15 min, then 850 mg of PS was introduced in the mixture, and the reagents were allowedto react for 45 min at room temperature. Carbon disulfide  was introduced and allowed to reactfor a further 15 min. Ethyl α-bromophenylacetate  was added and reacted overnightat room temperature; 703 mg (yield 65 wt %) of a yellow oily materialwas obtained.1H NMR (CDCl3): δ= 1.22–1.32 –CH(Ph)–S−),1.35–1.45 , 2.55–2.70, 2.71–2.78, 2.85–3.05, 3.61–3.68, 4.11–4.35 –CH(Ph)–S−),5.77–5.81 –CH(Ph)–S−),7.33–7.47 ppm .13C NMR (CDCl3): δ = 14.1 (CH3–CH2–O–C(=O)–CH(Ph)–S−),20.8 (CH3 in PPS chain), 32.4 (−S–CH2–CH2–O–CH2–CH2–O–CH2–CH2–S−),38.4 (CH2 in PPS chain), 41.2 (CH in PPS chain), 48.4 (CH3–CH2–O–C(=O)–CH(Ph)–S−),61.9 (CH3–CH2–O–C(=O)–CH(Ph)–S−),70.4 (−S–CH2–CH2–O–CH2–CH2–O–CH2–CH2–S−), 71.2 (−S–CH2–CH2–O–CH2–CH2–O–CH2–CH2–S−), 128.8 (phenyl carbonin para position), 128.9 (phenyl carbons in ortho position), 129 (phenyl carbons in meta position), 133 (phenyl carbon in ipso position),168.7 (CH3–CH2–O–C(=O)–CH(Ph)–S−),220.9 ppm (−CH(Ph)–S–C(=S)–S–CH2−).as CH3), 2919 (νas CH2), 2865 (νs CH3 and νs CH2), 1736(νas C=O), 1370 (νas C–O–C),820 (νas C=S), 734 cm–1 (νas C–S).FT-IR (ATR): ν̅ = 2958 : M̅n = 3600 g/mol, Đ = 1.01.MALDI-ToF(dithranol matrix without cationizing agent at 1:1 polymer-to-matrixratio in THF): DOTAc , TBP ,and 1.2 mL  of a 0.5 M sodiummethoxide solution in methanol were added in 5 mL of THF. The mixturewas stirred for 15 min. Acetic acid  and DBU were added followed by 850 mg of PS, and the reagents were allowed to react for 45 min at room temperature.Carbon disulfide  wasintroduced and allowed to react for a further 15 min. Ethyl 2-bromopropionate was added and reactedfor 5 h at room temperature; 717 mg (yield: 70 wt %) of a yellow oilymaterial was obtained.1H NMR (CDCl3):δ = 1.27–1.32 –CH(CH3)–S−),1.35–1.45 –CH(CH3)–S−), 2.57–2.74 , 2.74–2.79 , 2.85–3.01 , 3.61–3.68 ,4.18–4.32 –CH(CH3)–S−), 4.77–4.85 ppm –CH(CH3)–S−).13C NMR (CDCl3): δ = 14.1 (CH3–CH2–O–C(=O)–CH(CH3)–S−), 20.8 (CH3 in PPS chain andCH3–CH2–O–C(=O)–CH(CH3)–S−), 32.4 (−S–CH2–CH2–O–CH2–CH2–O–CH2–CH2–S−),38.4 (CH2 in PPS chain), 41.2 (CH in PPS chain), 48.4 (CH3–CH2–O–C(=O)–CH(CH3)–S−), 61.9 (CH3–CH2–O–C(=O)–CH(CH3)–S−),70.4 (−S–CH2–CH2–O–CH2–CH2–O–CH2–CH2–S−), 71.2 (−S–CH2–CH2–O–CH2–CH2–O–CH2–CH2–S−), 168.7 (CH3–CH2–O–C(=O)–CH(CH3)–S−), 220.9 ppm (−CH(CH3)–S–C(=S)–S–CH2−).as CH3),2919 (νas CH2), 2865 (νs CH3 and νs CH2), 1736(νas C=O), 1370 (νas C–O–C),820 (νas C=S), 734 cm–1 (νas C–S).FT-IR(ATR): ν̅ = 2958 : M̅n = 2900 g/mol, Đ = 1.02.MALDI-ToF(dithranol matrix without cationizing agent at 1:1 polymer-to-matrixratio in THF): DOTAc , TBP , and 1.2 mL  ofa 0.5 M sodium methoxide solution in methanol were added in 5 mL ofTHF. The mixture was stirred for 15 min. Acetic acid  and DBU  were added followed by 850 mg  of PS, and the reagents were allowed toreact for 45 min at room temperature. Ethyl 2-bromopropionate  was added and reactedovernight at room temperature; 583 mg (yield: 53 wt %) of a yellowoily material was obtained.1H NMR (CDCl3): δ = 1.27–1.32 –CH(CH3)–S−),1.35–1.45 –CH(CH3)–S−), 2.57–2.74 , 2.74–2.79 , 2.85–3.01 , 3.46–3.57 –CH(CH3)–S−), 3.61–3.68 ,4.18–4.32 –CH(CH3)–S−), 4.77–4.85 ppm –CH(CH3)–S−).13C NMR (CDCl3): δ = 14.1 (CH3–CH2–O–C(=O)–CH(CH3)–S−), 20.8 (CH3 in PPS chain andCH3–CH2–O–C(=O)–CH(CH3)–S−), 32.4 (−S–CH2–CH2–O–CH2–CH2–O–CH2–CH2–S−),38.4 (CH2 in PPS chain), 41.2 (CH in PPS chain), 48.4 (CH3–CH2–O–C(=O)–CH(CH3)–S−), 61.9 (CH3–CH2–O–C(=O)–CH(CH3)–S−),70.4 (−S–CH2–CH2–O–CH2–CH2–O–CH2–CH2–S−), 71.2 (−S–CH2–CH2–O–CH2–CH2–O–CH2–CH2–S−), 168.7 (CH3–CH2–O–C(=O)–CH(CH3)–S−).as CH3), 2919 (νas CH2), 2865 (νs CH3 and νs CH2), 1736(νas C=O), 1370 (νas C–O–C),734 cm–1 (νas C–S).FT-IR (ATR): ν̅ = 2958: M̅n = 3100 g/mol, Đ = 1.01.MALDI-ToF (dithranol matrix withoutcationizing agent at 1:1 polymer-to-matrixratio in THF): 1H NMR on nonpurified samplesby normalizing to the trioxane at 5.1 ppm and measuring the consumptionof monomer via the loss of the peak at 5.6 ppm. The polymerizationswere stopped by exposure to air and placing on ice when monomer consumptionreached a plateau. The reaction solution was diluted in deionizedwater followed by dialysis and freeze-drying. The purified block copolymerswere characterized by 1H NMR and GPC.All experimentswere performed in a 12-position Carousel parallel reactor. In a typicalpolymerization, PPS-macroRAFT agent (PMA), AIBN, monomer, and 5 mg/mLof trioxane as an internal standard were dissolved in dry DMF. Themixture was degassed with argon for 45 min and reacted at 70 °Cunder argon atmosphere. Samples were collected during the reactionsfor conversion analysis via BB-PMA , AIBN , DMA, and 5 mg/mL of trioxaneas an internal standard were dissolved in 6 mL of dry DMF and reactedfor 8 h; 648 mg (yield: 93 wt %) of white fluffy material was obtained.1H NMR (CDCl3): δ = 1.05–1.27, 1.27–1.37, 1.38–1.64 , 1.64–1.91 , 2.15–2.72–N(CH3)(CH3))]n–S–C(=S)–;CH in PDMA chain; t, SCH2CH2OCH2CH2OCH2CH2S–; and 1 diastereotopicCH2 in PPS chain), 2.71–3.16 , 3.53–3.63 ,7.03–7.15 , 7.15–7.22 ppm .M̅n = 10600 g/mol, Đ = 1.15.GPC (DMF): BPA-PMA (200 mg0.054 mmol), AIBN , DMA, and 5 mg/mL of trioxaneas an internal standard were dissolved in 6 mL of dry DMF and reactedfor 78 h; 507 mg (yield: 80 wt %) of white fluffy material was obtained.1H NMR (CDCl3): δ = 1.05–1.27–CH(Ph)−),1.27–1.37 , 1.38–1.64, 1.64–1.91, 2.49–2.72,2.71–3.16 , 3.53–3.63–CH(Ph)−), 3.91–4.10ppm –CH(Ph)−).M̅n = 11800 g/mol, Đ = 1.08.GPC (DMF): BP-PMA , AIBN , DMA , and 5 mg/mL of trioxane as aninternal standard were dissolved in 6 mL of dry DMF and reacted for5 h; 751 mg (yield: 92 wt %) of white fluffy material was obtained.1H NMR (CDCl3): δ = 1.05–1.27–CH(CH3)−); and t, CH3–CH2–O–C(=O)–CH(CH3)−), 1.27–1.37 ,1.38–1.64 , 1.64–1.91 , 2.21–2.72 –CH(CH3)–S−);and CH in PDMA chain), 2.71–3.16 ,3.53–3.63 , 3.91–4.10ppm –CH(CH3)−).M̅n = 13700 g/mol, Đ = 1.18.GPC (DMF): BP-PMA , AIBN , 5 mg/mLof trioxane as an internal standard, and monomers were dissolved in6 mL of dry DMF and reacted for 5 h. Monomer quantities and yields:(A) 100% DEA, 744 mg DEA ; yield 869mg (79 wt %). (B) 75:25 DEA/DMA, 145 mg of DMA, 558 mg  of DEA; yield 840mg (92 wt %). (C) 50:50 DEA/DMA, 290 mg of DMA, 372 mg  of DEA; yield 776mg (90 wt %). (D) 25:75 DEA/DMA, 435 mg of DMA, 186 mg  of DEA; yield 752mg (97 wt %).1H NMR (CDCl3): δ= 0.87–1.27 –CH(CH3)–; d, 6H, CH3–CH2–O–C(=O)–CH(CH3)–; CH3 in PDEA chain; and CH2 in PDEA and PDMA chains, m diads), 1.27–1.37, 1.38–1.64 , 1.64–1.91,2.21–2.72 –CH(CH3)–S–; DEA and DMA chain CH), 2.72–3.54, 3.54–3.63 , 3.91–4.10ppm –CH(CH3)−).GPC (DMF): please refer to l-glutamine at 37 °C in a humidified atmosphereof 5% CO2. BV2 microglia and C8D30 astrocytes were culturedin DMEM-high glucose (4500 mg/L glucose) supplemented with 10% FBS,100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate,and 2 mM l-glutamine at 37 °C in a humidified atmosphereof 5% CO2. Cell culture maintenance was performed by washingthe cells with Dulbecco’s phosphate-buffered saline (PBS),followed by cell detachment using trypsin. The detached cells werethen suspended in culture medium, counted using trypan blue dye, platedin culture plates, and cultured at 37 °C.RAW 264.7 macrophages and fibroblastswere cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin,100 μg/mL streptomycin, 0.25 μg/mL amphotericin B, and0.2 M in vivo experiments were performedin accordance with the guidelines and regulations of the EuropeanDirective 2010/63/EU and approved by the Italian Ministry of Health(authorization code: 22418.N.KOL). Pregnant mice (C57BL/6J) were sacrificed,and the uterus was dissected out and placed in a sterile Petri dishwith HBSS. Embryonic sacs were removed from uterus and transferredto a 6 cm HBSS-filled dish. Using a dissecting microscope, the brainsof the embryos and then the cortices of those embryos were dissectedout and were placed in a dish containing HBSS. Cortices were cut intosmall pieces and washed 3 times with HBSS. Ten milliliters of 0.25%trypsin was added and incubated for 25–30 min in a water bathat 37 °C. The solution was discarded, and the cortices were washedtwice with HBSS followed by addition of Neurobasal medium supplementedwith 5% FBS. The merged cell suspensions were filtered through a 40μm filter. The cell culture plates were coated with 0.1 mg/mLpoly(l-lysine) (PLL) overnight, washed with deionized waterthe day of the dissection, and dried. Cells were cultured in Neurobasalmedium supplemented with 2% B-27, 100 U/mL penicillin, 100 μg/mLstreptomycin, and l-glutamine at 37 °C in a humidifiedatmosphere of 5% CO2.Primary culturesof mouse cortical neurons were prepared from embryos on day 17–18of pregnancy, and all 4 cells per well for fibroblastsand macrophages, 2 × 104 cells per well for BV2, and3 × 104 cells per well for C8D30) and allowed to adhereovernight. Cells were washed with PBS, and 200 μL of fresh mediumwith polymers at a concentration of 0.01, 0.05, 0.1, 0.5, 1, 5, and10 mg/mL was added. After 24 h incubation, the medium was discarded,cells were gently washed with PBS and incubated for 1 h at 37 °Cwith CellTiter 96 Aqueous One Solution Reagent in medium preparedby following the manufacturer instructions, and the absorbance wasmeasured at 490 nm using a plate reader. The solution was then removed,and cells were washed with PBS. To lyse the cells, 100 μL ofRIPA buffer was added; the cells were kept −80 °C overnightfollowed by incubation at 37 °C for 1 h. The total protein contentwas determined using Pierce BCA Protein Assay Kit following the manufacturerinstructions as follows: 25 μL of lysed cell solution was mixedwith 200 μL of BCA working reagent, incubated at 37 °Cfor 30 min, and cooled to room temperature, and the absorbance wasmeasured at 562 nm using a plate reader. The absorbance readings ofthe treated cells obtained from CellTiter 96 Aqueous One SolutionAssay and Pierce BCA Protein Assay were normalized to the absorbancereading of the untreated cells. The ratio between the metabolic activity(%) and the protein content (%) was determined, and the results areexpressed as the percentage of treated vs untreated cells.For metabolic activity assay, the cellswere seeded in 96-well plates (10Metabolic activity was assessed viaa generic metabolic activity assay (MTT), and via a live–deadassay.4 cells per PLL-coated well (seeabove) of a 96-well plate, keeping them in culture (200 μL ofmedium) for 6 days. Then 100 μL of medium was removed, and 100μL of polymer dispersions in medium at concentrations of 0.02,0.1, 0.1, 1, and 2 mg/mL was added, to obtain final polymer concentrationsof 0.01, 0.05, 0.1, 0.5, and 1 mg/mL, respectively. Cells were finallyincubated for 24 and 48 h, and CyQUANT MTT Cell Viability Assay wasperformed following the manufacturer instructions with appropriateadaptations as follows: 100 μL of the medium was discarded,10 μL of MTT stock solution was added, and the cells were incubatedat 37 °C for 4 h followed by addition of 100 μL of SDS-HClsolution and further incubation at 37 °C for 4 h. The absorbancewas measured at 570 nm. The results are expressed as the percentageof treated vs untreated cells.Primary cortical neurons were seeded at adensity of 2 × 104 cells per well on glass coverslips (12 mmdiameter) that were precoated with 0.1 mg/mL of PLL (100 μL/well)on a 24-well plate (1 mL of medium) and kept in culture for 6 days.Then 500 μL of medium was removed and 500 μL of polymerdispersions in the medium at concentrations of 0.02, 0.1, 0.1, 1,and 2 mg/mL was added, to obtain final polymer concentrations of 0.01,0.05, 0.1, 0.5, and 1 mg/mL, respectively. After 24 h incubation,1 μL of propidium iodide, calcein-AM, and Hoechst dye solutionswas added. All nuclei , live cells , and dead cells  were visualized under a NIKON Eclipse Ti epifluorescence microscope using NIS-Elements imaging software andImageJ for the image analysis; 3–5 random fields were imagedper culture well. The average metabolic activity per cell was calculatednormalizing the MTT data against the number of attached (DAPI-positive)cells.Neurons were seeded at a density of3 × 104 cells per well and allowed toadhere overnight. The following day, cells were treated for 24 h with0.5 μg/mL LPS in fresh DMEM before an additional 24 h incubationwith DMEM containing 0.5 μg/mL LPS and 0.5 and 1 mg/mL polymers.Control groups consisted of untreated cells and LPS-stimulated cellswith no polymer treatment. Culture supernatants were collected andcentrifuged at 1000 rpm for 5 min and analyzed for the presence ofTNF-α by ELISA, as well as for the measurement of LDH levels.BCA assay was performed to quantify the total protein content perwell.Cells were seeded on 96-well plates ata density of 2 × 105 cells per well on a 24-well plate andallowed to adhere overnight. The following day, BV2 cells were treatedfor 24 h with 0.5 μg/mL LPS in fresh DMEM. The same day C8D30astrocytes were seeded at a density of 3 × 104 cellsper well in a 96-well plate and allowed to adhere overnight. On thenext day, the supernatants of BV2 cells were collected and centrifugedat 1000 rpm for 5 min to remove the dead cells and debris. MCM wasprepared by mixing 80% (v/v) of the supernatant collected after centrifugationwith 20% (v/v) of fresh DMEM. The culture medium of C8D30 cells wasreplaced with 200 μL per well of MCM, and cells were incubated24 h before an additional 24 h incubation with 0.5 or 1 mg/mL polymersin fresh MCM. Control groups consisted of untreated cells and MCM-stimulatedcells with no polymer treatment. Supernatants were collected and analyzedas described above.These cells were activated using conditionedmedium (MCM) from LPS-stimulated BV2 cells. BV2 were seeded at a densityof 1.5 × 104 cells per well on a 96-well plate (precoatedwith 0.1 mg/mL of PLL (200 μL/well), keeping them in culture(200 μL of medium) for 6 days. Then 100 μL of medium wasremoved, 100 μL of MCM per well was added, and cells were incubatedfor 24 h before an additional 24 h incubation with polymers (0.5 and1 mg/mL) in fresh MCM. Control groups consisted of untreated cellsand MCM stimulated cells with no polymer treatment. Supernatants werecollected and analyzed as described above.Primaryneurons were activatedusing conditioned medium (MCM) from LPS-stimulated BV2 cells obtainedas described above. Primary neurons were seeded at a density of 2× 10TNF-αlevels were quantified by ELISA using mouse kits following the manufacturer’sinstructions. Values were normalized against the total protein contentper well, setting a 100% value for the level of membrane damage obtainedby treating cells with MCM alone.Membrane damage aftertreatments was analyzed using an LDH assay kit according to the manufacturer’sinstructions. In brief, supernatants were collected, LDH was measured,and optical densities were normalized against the total protein contentper well, setting a 100% value for the level of membrane damage obtainedby treating cells with MCM alone.p < 0.05,**p < 0.01, ***p < 0.001,****p < 0.0001. Statistical analyses were conductedusing GraphPad Prism 9.3.1Data are presented as mean values± standard error of the mean (SEM). Normality was assessed withthe Shapiro–Wilk test. The statistical analysis employed one-wayanalysis of variance (ANOVA) test with Dunnett’s post hoc testor the Kruskal–Wallis test with Dunn’s post hoc test.Accepted levels of significance were *Supporting Information, section 1.1SI and Figure S1), whose thioacetates were first converted into active sodium thiolatesvia reaction with sodium methoxide. This procedure, together withthe use of an in situ reducing agent such as tributylphosphine(TBP), aims to minimize the presence of disulfides in the polymerizationenvironment, which would act as transfer agents in the episulfideROP, causing the formation of polydisperse and/or wrongly end-cappedpolymers.33 The episulfide used as a monomer in thisstudy  was also synthesized, using a scaled-upand automatized procedure .Here, we have employeda protected dithiol initiator , four secondary , and a tertiary bromide .From consensus guidelines,34 it is expectedthat the transfer coefficients of these groups will decrease in theorder BPA > BIB ≫ BPN > BP ≈ BPAM > BB ≳polysulfidechain. Indeed, a secondary radical-terminated PPS is less stable thanany of the radicals  possiblyproduced by this set of end-cappers.Upon additionof PS to the deprotected thiolate initiator, polymerization readilyensued; after 45 min CSsection 1.3SI and Table S1), with the aim to obtain quantitative end-cappingand avoid multimodal molecular weight distribution, which may arisefrom uncapped PPS thiolates. As a result of this phase, well-definedpoly(propylene sulfide) macroRAFT agents (PMAs) could only be producedusing BB, BPA, and BP .We have screened severalreaction conditions to produce these trithiocarbonatederivatives  was produced under the same reaction conditions used forthe synthesis of BP-PMA, minus the use of CSgreement 1D.13C NMR and an IR absorption at 820 cm–1 attributedto C=S groups. The near UV absorption of trithiocarbonatesat 310 nm in principle can provide more quantitative information,but it is critical to choose a model compound with substituents similarto the target group. For example, the fully aliphatic bis(carboxymethyl)trithiocarbonate)is structurally similar to the trithiocarbonate in BP-PMA, and usingthe extinction coefficient of the former, the latter indeed appearsto bear two C=S groups per chain, i.e., quantitative end-capping. BB-PMA and BPA-PMA, on the contrary, feature phenylrings close to the trithiocarbonate group, which is likely differentin extinction coefficient. However, since the reaction between PPSterminal thiols and CS2 was performed under identical conditionsfor all PMAs, we assumed them to be all quantitatively end-capped.Qualitatively, the incorporation of trithiocarbonatesin the PMAswas confirmed through the presence of a resonance at 220.9 ppm in 1H NMR as a function of time. With BB-PMA (Đ = 1.1–1.2), as observed in 1H NMR spectroscopy.Finally, polymerization in the presence of BP-NEG, on the other hand,led to a more rapid monomer consumption  was employed to screen the three PMAs describedabove. In all experiments, DMA conversion was detected by h BB-PMA 2A, top w(A)The character of the DMA polymerizationwith BP-NEG is quite different from the others and did not lead toappreciable integration of DMA with the polysulfide chains, as onthe contrary was observed for the other PMAs. In short, we can confirmthat the latter do polymerize with a RAFT and not a free radical mechanism.(B)The inhibition periodwas considerablylonger in BPA-PMA than in BB-PMA and BP-PMA. This seems to suggestthat the overall behavior of these PMAs is ascribed to the natureof the leaving radicals.We notethe following:34 due to itsstability. As a result, we chose BP-PMA for all further polymerizations,because of its higher monomer conversion rate.We ascribe thelong inhibition period of BPA-PMA to the low reactivity/reinitiationrate of the leaving BPA radical, which indeed is not one of the mostcommonly used RAFT agents,35 Importantly, CAC values were independent ofthe DMA/DEA ratio, which shows the aggregation to be solely influencedby the PPS block, at least at room temperature. Of note, PDIs arerelatively high, although below what typically is defined as polydisperse;36 this may relate to the presence of some larger(and out-of-equilibrium) aggregates, but their large values are alsodue to the small size of these colloids: the peak half-widths are<40 nm for all polymers bar PSE.In water, all these amphiphilictriblock polymers produced colloidal structures with a size significantlybelow 100 nm  it was possible to see well-defined cloud points in the region37–42 °C, which indicate the formation of much largeraggregates; at even higher concentrations, macroscopic gelation wasobserved .In short, at the cell culture/body temperature of 37 °C, PSEformed large, micrometer-sized aggregates (likely with poorly hydratedhydrophilic coronas), while all other polymers assembled in <100nm colloids. It is noteworthy that the extent of PSE aggregation markedlydepended on concentration. For example, at 1 mg/mL a transition canonly be recorded via DLS  determines their overall morphology: stablemicelles for PSM and PSM3E1, large aggregates for PSE, and likelydynamic micelles (close to an aggregation transition) for the intermediatePSM1E1 and PSM1E3l.To ascertain whether these morphologicaldifferences may impactcell viability, we have employed four well-established cell lines,monitoring the number of attached cells  cells and reduced that of calcein-positive ones for concentrations≥0.5 mg/mL. Curiously, the neuron mitochondrial activity remainedlargely untouched, despite the extensive membrane damage  and the loss of active cytoplasmic enzymatic activity. This behavior, quite different from, e.g., that of therather similar PSM1E3, may be due to PSE being the only polymer actuallyproducing pretty large aggregates, which would sediment on top ofthe cells, both creating a physical barrier to nutrient diffusionand artificially increasing the local concentration of polymer. Thisindicates that thermal aggregation may actually lead to detrimentaleffects, e.g., limiting the advantageous performance of PPS-basedmaterials as demonstrated in the CNS.caveat inmind, we have evaluated theROS scavenging-based anti-inflammatory activity, using three cellularmodels of neuroinflammation: LPS-activated microglia and astrocytesand neurons activated using microglia-conditioned medium, MCM, whichallows one to avoid the direct use of the rather toxic LPS on thesemore sensitive cells.39With this (A)In these three cellular models ofCNS inflammation, activation coincides with the release of the pro-inflammatorycytokine TNF-α (graphs in the left part of (B)At 1 mg/mL, PSE was detrimental: itincreased the release of LDH in activated astrocytes and microgliaand in the latter also increased TNF-α production (white arrowsin (C)Treatment with any other polymer (0.5or 1 mg/mL) did not induce any further LDH release, and at 1 mg/mLalmost always significantly reduced the release of TNF-α  aggregation at the cellculture temperature appears to be detrimental to delicate cells suchas primary cortical neurons. A decrease in viability is also apparentfor ≥0.5 mg/mL PSE  by exposed DEAresidues, although we cannot rule out an effect on, e.g., oxygen andnutrient permeation by the aggregates sedimenting on the cell layers.Of note, the latter effects may be specific to CNS cells: the lackof toxicity on 3T3 fibroblasts and RAW macrophages may suggest applicabilityto other less sensitive loci within the body.In summary, using a panel of cells that recapitulates thecellulardiversity of the CNS, this family of polymers showed remarkably lowtoxicity and promising anti-inflammatory activity, above all on inflamedneurons. The main g/mL PSE 6 and 7, g/mL PSE 5; this aIn this study, we have optimized the synthetic conditions to obtainwell-defined oxidation-sensitive amphiphilic block copolymers viasuccessive anionic and RAFT polymerization. This has allowed us toprepare a family of polymers virtually identical in primary structure,molecular weight distribution, and micellization behavior but differingin the composition of the hydrophilic shell. This parameter (the DEA/DMAratio) affected the kinetics of the oxidative response: the more hydrophilic,the more rapid the response, although the differences were not hugeand indeed all polymers exhibited anti-inflammatory behavior on threedifferent activated cellular models.in situ formation of localizeddepots at specific body locations upon injection of appropriatelycold formulations. On the other hand, although this family of polymersshowed no toxicity in classical cell lines (3T3 fibroblasts and RAWmacrophages) and limited effects in astrocytes and microglial celllines , the thermoresponsive, depot-formingPSE (PDEA-based) presented clear toxicity in clinically relevant primarycortical neurons.The hydrophile compositionhad a more dramatic effect on the capacityof micellar dispersions to undergo large-scale aggregation: only high-DEApolymers showed temperature-induced transitions potentially leadingto a macroscopic aggregation. This feature, on the one hand, is potentiallybeneficial allowing the Although this effect may be affected by theenvironmental constraintsof 2D cultures (sedimentation of aggregates), it highlights the necessityof exercising caution in the use of depot-forming systems for braindelivery. Having said this, all other micellar polysulfides, not aggregatingat physiological temperatures, were capable of blocking neuroinflammationin microglia, astrocytes, and primary cortical neurons, which makesthem attractive for further biomedical investigations.


Patient-derived triple-negative BCSCs are shown to be efficiently recognized and killed by γδ T cells, but xenografted BCSCs evade immune recognition by γδ T cells by various mechanisms, paving the way for novel combinatorial immunotherapies for TNBC. ex vivo expanded γδ T cells from healthy donors. Orthotopically xenografted BCSCs, however, were refractory to γδ T-cell immunotherapy. We unraveled concerted differentiation and immune escape mechanisms: xenografted BCSCs lost stemness, expression of γδ T-cell ligands, adhesion molecules, and pAgs, thereby evading immune recognition by γδ T cells. Indeed, neither promigratory engineered γδ T cells, nor anti–PD-1 checkpoint blockade, significantly prolonged overall survival of tumor-bearing mice. BCSC immune escape was independent of the immune pressure exerted by the γδ T cells and could be pharmacologically reverted by zoledronate or IFNα treatment. These results pave the way for novel combinatorial immunotherapies for TNBC.There are no targeted therapies for patients with triple-negative breast cancer (TNBC). TNBC is enriched in breast cancer stem cells (BCSC), which play a key role in metastasis, chemoresistance, relapse, and mortality. γδ T cells hold great potential in immunotherapy against cancer and might provide an approach to therapeutically target TNBC. γδ T cells are commonly observed to infiltrate solid tumors and have an extensive repertoire of tumor-sensing mechanisms, recognizing stress-induced molecules and phosphoantigens (pAgs) on transformed cells. Herein, we show that patient-derived triple-negative BCSCs are efficiently recognized and killed by  In breast cancer, which was the most commonly diagnosed cancer worldwide in 2020, breast cancer stem cells (BCSC) play a key role in metastasis formation, tumor recurrence, and mortality of patients . Specifiex vivo engineered αβ T cells back into patients have recently been employed for treating a variety of cancers. However, the efficacy of these treatments relies on the presence of tumor-specific antigens presented on MHC I molecules. The loss of MHC I expression and the concomitant lack of peptide presentation by cancer cells can undermine conventional αβ T-cell target recognition, and thus lead to tumor immune escape, which has been widely observed in cancers, including TNBC (Immunotherapies involving the transfer of autologous ing TNBC . To overing TNBC .+ T cells) and Vδ1+ γδ T cells (Vδ1+ T cells). Vδ2+ T cells represent the predominant γδ T-cell subset in the human blood and specifically recognize pAgs –expressing γδ T cells , the major noncellular fraction of the tumor microenvironment, negatively affects the migration and infiltration of αβ T cells in non-small cell lung cancer and ovarian cancer . High stWe sought here to use a model aiming to reflect the situation in human patients with TNBC to test the effect of γδ T cell–based therapy.2. Cells were regularly tested for Mycoplasma but were not reauthenticated.Healthy human dermal fibroblasts of neonatal origin  were cultured in DMEM GlutaMAX medium  supplemented with 10% FBS , 100 μg/mL penicillin, 100 μg/mL streptomycin , and 10 mmol/L HEPES . HEK293T cells  were grown in DMEM GlutaMAX medium supplemented with 10% FBS, 100 μg/mL penicillin, 100 μg/mL streptomycin, 10 mmol/L HEPES, and 10 μmol/L sodium pyruvate . HDFn and HEK293T cells were cultured at 37°C in a humidified atmosphere of 7.5% CO2, 5% CO2, 92% N2). Three-dimensional (3D) cells stably proliferating were cultured and expanded in two-dimensional MSC medium. Passaging of BCSCs was performed as described previously .BCSC generation has been described previously . Brieflyeviously . Experim6 cells/mL in γδ T-cell medium: RPMI1640 medium  supplemented with 10% FBS, 100 μg/mL penicillin, 100 μg/mL streptomycin, 10 mmol/L HEPES, 10 μmol/L sodium pyruvate, and 1× MEM nonessential amino acids . Expansion was induced with 1 μg/mL concanavalin A , 10 ng/mL IL2 , and 10 ng/mL IL4 . Cells were adjusted every 3 to 4 days to 1 × 106 cells/mL with γδ T-cell medium and cytokines. The cells were cultured at 37°C in a humidified atmosphere of 5% CO2. At days 10 to 14 after expansion started, γδ T cells were separated from αβ T cells via negative selection . Cultures with a purity of ≥ 95% were used for experiments from day 14 on.Primary γδ T-cell expansion was performed as described previously . BrieflyMMP14 cDNA or the cDNA of the catalytically inactive form of MMP14 E240A were cloned into the lentiviral backbone pLVX-CMV-IRES-zsGreen1 (Takara/Clontech #632187) by Gibson assembly. The CMV promoter was exchanged with a short EF-1α promoter sequence(GATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTGTCGTGACGCG). The integrity of each plasmid was verified by restriction enzyme digestion and Sanger sequencing.7 HEK 293T cells were plated on 15 cm dishes and cultured at 37°C and 7.5% CO2. After 24 hours, the medium was exchanged and HEK293T cells were transfected with the indicated constructs and the packaging plasmids pCMV-dR8.74 and pMD2-vsvG  using polyethylenimine transfection . After 24 and 48 hours, the viral particle-containing supernatant was harvested, pooled, filtered, and concentrated using density centrifugation [10% sucrose w/v in PBS/0.5 mmol/L Ethylenediaminetetraacetic acid (EDTA)] for 4 hours at 10,000 × g and 6°C. The supernatant was discarded, and the viral particles were resuspended in PBS using 1/400th of the harvested volume and stored at −80°C.For the generation of lentiviral particles, 1 × 10γδ T cells were lentivirally transduced with a multiplicity of infection (MOI) of 2-5 as indicated for the individual experiments. Transduced γδ T cells were checked for zsGreen1 and matrix metalloprotease 14 (MMP14) expression by flow cytometry after 2 to 3 days after transduction.To stain cell surface proteins, cells were washed once with flow cytometry buffer (PBS supplemented with 2% FBS) and incubated in the diluted antibody solution for 15 minutes at 4°C. In the case of fluorophore-labeled antibodies, cells were washed once with flow cytometry buffer and analyzed on a Gallios flow cytometer (Beckman Coulter). The flow cytometry results were analyzed using FlowJo v10.8 Software (BD Life Sciences). Unlabeled primary antibodies were visualized using fluorescently labeled secondary antibodies. After washing away the primary antibody, cells were incubated in the diluted secondary antibody solution for 15 minutes at 4°C. Finally, cells were washed once as described above and analyzed on a Gallios flow cytometer.Self-made anti-CD3ε (clone UCHT1) and anti-CD28 (clone CD28.2) from BioLegend were used for γδ T-cell stimulation. Monoclonal anti-HMGCR  and anti-Vinculin  were used for immunoprecipitation (IP) and Western blot analysis.+ T Cell Isolation Kit to separate Vδ2+ and Vδ1+ cells from γδ T-cell expansion cultures, respectively.For flow cytometry, the following antibodies were used: anti-EpCAM-AlexaFluor488 (clone 9C4), anti-CD107a-PE (clone H4A3), anti-BTN3A-PE (clone BT3.1), anti-Fas-BV421 (clone DX2), anti-TRAILR1-APC (clone DJR1), anti-TRAILR2-PE (clone DJR2-4), anti-ICAM-1-BV421 (clone HA58), anti-CD3ε-AlexaFluor488 (clone UCHT1), anti-CD3ε-AlexaFluor647 (clone UCHT1), anti-CD27-PE (clone M-T271), anti-NKG2D-APC (clone 1D11), anti-LAG-3-AlexaFluor647 (clone 11C3C65), anti-PD-L1-APC (clone 29E.2A3), anti-PD-L2-BV421 (clone 24F.10C12), anti-Vδ2TCR-Biotin (clone B6), anti-CXCR1-APC (clone 8F1),  anti-CXCR3-AlexaFluor647 (clone G025H7), anti-CXCR4-PE (clone 49801),  anti-CXCR5-APC (clone J252D4), anti-CXCR6-PE (clone K041E5), α-CCR2-APC/Fire 750 (clone K036C2),  anti-CCR3-FITC (clone 5E8), anti-CCR10-APC (clone 6588-5), and anti-TIM-3-PE-Cy7 (clone F38-2E2) from BioLegend.  Anti-ULPB2/5/6  and  anti-CCR4-APC (clone 205410) from R&D Systems. Anti-mouse IgG-APC from Southern Biotech. Anti-γδTCR-PE (clone SA6.E9), anti-γδTCR-FITC (clone SA6.E9), anti-rabbit-DyLight633 , anti–PD-1-PE-Cy7 (clone J105), streptavidin-eFluor450 (The following antibodies were used for immunofluorescence: α-EpCAM-BV421 (clone EBA-1) from BD Biosciences, α-fibronectin  and α-rabbit IgG-AlexaFluor546  from Sigma-Aldrich. CellTracker Green CMFDA Dye was used from Thermo Fisher Scientific (C7025).The following antibodies were used for blocking experiments: anti-Fas (clone A16086F), anti-FasL (clone NOK-1), anti-TRAIL (clone RIK-2), anti-TRAILR1 (clone DJR1), anti-TRAILR2 (clone DJR2-4), anti-NKG2D (clone 1D11), anti-MICA/B (clone 6D4), anti-ICAM-1 (clone HCD54), anti-IgG1 isotype (clone MOPC-21), and anti-IgG2b isotype (clone MG2b-57) from BioLegend. Anti-ULPB2/5/6 (clone 165903) from R&D Systems. Anti-CD103 (clone 2G5) from Beckman Coulter. Zoledronic acid monohydrate  and mevastatin (M2537) were purchased from Sigma-Aldrich.Viability of γδ T cells was assessed using the FITC Annexin V Apoptosis Detection Kit I .4 luciferase-expressing target cells were plated in a white 96-well flat-bottom plate. Effector cells were added to the target cells at the desired effector to target cell ratios. A total of 37.5 μg/mL d-Luciferin Firefly  was added to the samples, which were then incubated for the indicated time at 37°C and measured using a luminometer (Tecan infinity M200 Pro). Bioluminescence was measured as relative light units (RLU). RLU signals from target cells alone served as spontaneous death controls. Maximum killing RLUs were determined using target cells lysed with 1% Triton X-100. The specific lysis was calculated with the following formula:Bioluminescence-based cytotoxicity assays were performed as described previously . Briefly51Cr-release assays were performed to assess cytotoxicity in experiments including xenograft-derived tumor cells. Target cells (tumor cells) were loaded with 51Cr  for 1 hour at 37°C. After washing the cells three times with medium, cells were resuspended in γδ T-cell medium and 1 × 104 cells were plated in a 96-well U bottom plate. Effector cells (γδ T cells) were added to the target cells at the desired effector to target cell ratios. Samples were incubated for the indicated time at 37°C. Supernatants were transferred to a solid scintillator-coated 96-well plate  and then measured using a microplate scintillation γ-ray counter . Target cells without effector cells served as spontaneous 51Cr release controls. Maximum 51Cr release was determined using target cells lysed with 1:20 centrimide. The specific lysis was calculated with the following formula:Antibodies and chemicals).For blocking experiments, effector or targets cells were preincubated with 20 μg/mL of the indicated blocking antibodies at 37°C for 1 hour. IgG isotype antibodies were used as experimental controls. Cells were then used for cytotoxicity experiments in the presence of 10 μg/mL of the blocking antibodies detailed above  for 1 hour at 37°C. The assays were conducted with a final concentration of 1 × 103 U or 1 × 104 U IFNα 2B.To analyze the effect of IFNα 2B, target cells were preincubated with 2 × 105 tumor cells and 1 × 105 γδ T cells were cocultured for 3 hours at 37°C in the presence of 1 μL anti-CD107a-PE (BD Biosciences). Medium or stimulation with anti-CD3 and anti-CD28  served as negative and positive controls, respectively. Cells were harvested and analyzed by flow cytometry.To analyze γδ T-cell degranulation, BCSCs or xenograft-derived tumor cells were labeled with 20 μmol/L cell proliferation dye eFluor450 (Thermo Fisher Scientific). A total of 1 × 105 γδ T cells were cocultured with tumor cells for 24 hours at a ratio of 1:1. Target cells were preincubated with 20 μmol/L zoledronate for 2 hours or with 2 × 103 U or 2 × 104 U IFNα 2B for 1 hour at 37°C before coculturing them with γδ T cells. The assay was conducted with a final concentration of 10 μmol/L zoledronate or 1 × 103 U or 1 × 104 U IFNα 2B. The culture supernatant was then analyzed for secreted IFNγ using an IFNγ ELISA kit  according to the manufacturer's instructions.A total of 1 × 10NOD SCID mice  and Rag2-γ-mice  were housed at the Center for Experimental Models and Transgenic Service, Freiburg, under specific pathogen-free conditions using individually ventilated cages. Mouse handling and experiments were performed in accordance with German Animal Welfare regulations and approved by the Regierungspräsidium Freiburg .5 BCSCs were mixed with 1 × 106 irradiated HDF in a 1:1 mixture of MSC medium and Matrigel  and transplanted into each fat pad of the two #4 mammary glands of female NOD SCID mice. Mice were anesthetized during the procedure using an isoflurane inhalator. Before treatment started, mice were randomized into three groups: vehicle (PBS), γδ T cell, γδ T cell MMP14. Treatment was initiated for each mouse individually at a tumor volume of at least 4 mm3 as indicated for each experiment. A total of 5 × 106 γδ T cells  were intravenously injected three times per week for the indicated time period. In addition, mice received 0.6 × 106 IU of IL2  in incomplete Freund's adjuvant subcutaneously in the abdomen to support γδ T-cell survival in vivo. Checkpoint inhibition using the α-PD-1 antibody Nivolumab  and a corresponding α-human IgG4κ isotype control  was performed by additional biweekly intraperitoneal injections of 200 μg of the respective antibody. Tumor sizes were defined by caliper measurement. Tumor volumes were calculated using the formula V = 4/3 × π × r3.The orthotopic transplant was performed as described previously . A totalXenograft tumor model) were cut into small pieces using a razor blade and digested with 1 mg/mL collagenase IV  and 0.1 mg/mL DNAse I  at 37°C for 45 minutes. The digested tissue was filtered through 70 and 40 μm filters. To remove remaining cell debris, the cell suspension was cleared via centrifugation through a FBS layer. Erythrocytes were removed using Ammonium-Chloride-Potassium (ACK) lysis and recovered cells were used for experiments when the proportion of human EpCAM-positive (EpCAM+) cells was > 95%. For flow cytometric analysis, EpCAM+ cells were gated.Tumors from BCSC5 orthotopic xenografts  in PBS and 100 U/mL heparin  and centrifuged at 850 × g for 20 minutes at room temperature without break. Erythrocytes were removed from the pellet using ACK lysis and recovered cells were used for flow cytometry.Leukocytes were isolated from blood samples by repeated erythrocyte lysis steps using ACK lysis buffer. When only minimal residual erythrocytes were left in the sample, cells were used for flow cytometric analysis. To analyze liver-derived lymphocytes, liver tissue was cut into small pieces using scissors and further dissociated through a 70 μm filter. The cell suspension was centrifuged at 60 × Tumor slices were prepared as described previously . Briefly6 γδ T cells were labeled with 0.5 μmol/L CellTracker Green CMFDA Dye (Thermo Fisher Scientific) according to the manufacturer's instruction and mixed with an anti-rabbit-AlexaFluor564 (10 μg/mL). The solution was added on the tumor slices and incubated for 30 minutes at 37°C. Subsequently slices were washed and incubated at 37°C for 10 minutes until imaging.For fluorescent labeling of the tumor tissue and plating of γδ T cells, prewet stainless steel flat washers were placed onto the agarose surrounding each tumor slice and the slices were incubated at 37°C for 10 minutes. Then, tumor slices were stained with α-EpCAM-BV421 (10 μg/mL) and anti-fibronectin (3.5 μg/mL) for 15 minutes at 37°C and subsequently washed with slice assay medium. A total of 1 × 10Image acquisition was performed at 37°C in slice assay medium with a LSM 880 inverted laser scanning confocal microscope (Zeiss) equipped with a 25× objective  using the Fast Airyscan mode. BV421, CMFDA, and AlexaFluor546 were excited with a 405, 488, or 561 nm laser, respectively. Nine optical planes spanning a total depth of 63 μm in the Z dimension were captured every 30 seconds for 20 to 45 minutes.Z axis. Areas negative for fibronectin signal were considered as tumor regions. Further data analysis was performed using Python and the scikit-image library. Images were first corrected for sample drift by detecting matching features in subsequent frames of the ECM channel and estimating transformation parameters based on their coordinates. Cells were segmented by intensity after background correction to reduce bleed-through of signal from the ECM channel and median filtering for smoothing. Detected features were filtered by size to remove noise and cell clumps. To facilitate tracking, cells that were apparent in more than one Z plane were only considered in the plane in which their size was maximal. Subsequent tracking of the detected cells was performed using Trackpy, and two tracks were merged if their respective initial or final point were less than 10 μm and less than two frames apart. Only tracks with data for at least six frames were considered for further analysis.Airyscan data were first processed and stitched using the Zeiss Zen Black edition 3.0 SR. Then, ECM regions were manually defined with the help of the fibronectin staining in each individual plane along the For localization analysis, cells were considered to localize to tumor regions when no pixel of the γδ T-cell CMFDA signal overlapped with the fibronectin signal. A cell was defined to have a tumor dwell time >50% if it spent at least 50% of its observed frames localized to tumor regions. For speed analysis, the momentary speed of a cell was defined as the distance travelled between two successive observations of the same cell, divided by the time between these observations.Representative microscopy images and videos were generated using Imaris 9.3.1.For transwell migration assays, conditioned medium (CM) was generated by harvesting the supernatant of BCSC cultures after 5 days. Culture supernatants were cleared from residual cells by centrifugation and stored at −80°C until use.6 tumor cells were cultured in γδ T-cell medium for 24 hours at 37°C in a humidified atmosphere of 5% CO2. Culture supernatants were cleared from residual cells by centrifugation and directly used for experiments.To generate CM from xenograft-derived cells, 5 × 10For transwell migration assays toward CM, the CM was diluted 1:1.67 in γδ T-cell medium/1% FBS. A total of 250 μL were transferred to the receiver wells of a 96-well transwell plate (Corning). Medium without cells, incubated in the same conditions as BCSCs, served as negative control. For migration toward the chemokine CXCL12, γδ T-cell medium/1% FBS was supplemented with 50 ng/mL CXCL12 .5 tumor cells in 250 μL γδ T-cell medium/1%FBS were seeded into the receiver wells of a 96-well transwell plate. γδ T-cell medium/1% FBS served as negative control.For transwell assays toward tumor cells, tumor cells were labeled with 20 μmol/L cell proliferation dye eFluor450 (Thermo Fisher Scientific). A total of 1.5 × 105 γδ T cells in 100 μL γδ T-cell medium/1% FBS were seeded in the well of the permeable support with 5-μm pore size (Corning). After 3 hours incubation at 37°C, transmigrated cells were stained to distinguish T-cell subsets and counted via flow cytometry.A total of 2 × 104 γδ T cells were resuspended in 10 μL ice-cold Matrigel  and plated into the inner well of a precooled μ-Slide Angiogenesis (ibidi). After cell settling at 4°C, samples were solidified at 37°C. A total of 15 μL Matrigel were added on top of the cell-containing gels. Following solidification at 37°C, wells were filled with γδ T-cell medium supplemented with 10% FBS, 30 U/ml IL2, and 50 ng/mL CXCL12. After 48 hours, samples were fixed with 4% paraformaldehyde/0.25% glutaraldehyde for 20 minutes at room temperature. Gels were washed with PBS and cells were permeabilized with 0.5% Triton-X100 (Carl Roth) in PBS for 40 minutes. After washing, nuclei were stained with 5 μg/mL Hoechst 33342  for 3 hours in the dark and samples were imaged using confocal microscopy.Matrigel migration assays were performed as described previously . BrieflyImages were acquired using a C2 confocal microscope (Nikon) with a 20× objective . Hoechst 33342 was excited with a 405 nm laser. Z stacks with a step size of 2 μm were imaged. Quantification of the migration distance was performed by using Python and the scikit-image library. Briefly, the voxels of the stacks were segmented into foreground (cells) and background by intensity thresholding. Overlapping cells were then separated in 3D with the Watershed algorithm. The position of a cell was defined to coincide with its centroid. Migration distances were normalized between images by baseline subtraction per image: the baseline migration distance for an image was defined as the 10th percentile of all cells’ Z coordinates. This value was subtracted from all Z coordinates. Z coordinates were set to 0 if they became negative after baseline correction.2] and digested sequentially with LysC (Wako), then Trypsin , each at a 1:100 w/w (enzyme:protein) ratio. Peptide clean-up was performed according to the SP3 protocol.BCSC5 culture cells and xenograft-derived tumor cells were washed with PBS, pelleted, frozen in liquid nitrogen, and stored at −80°C until further processing. Cell pellets were lysed in 400 μL lysis buffer . Lysates were boiled for 5 minutes and then sonicated for 15 minutes at high intensity (30 seconds on/30 seconds off). After sonication, DNA and RNA were degraded using Benzonase endonuclease . The protein concentration was measured with EZQ Protein Quantitation Kit . Lysates were alkylated in the dark with 20 mmol/L iodoacetamide for 1 hour at room temperature. For protein clean-up, 200 μg SP3 paramagnetic beads were added to the lysates, and proteins were bound to the beads by adding acetonitrile with 0.1% formic acid. Beads were washed in 70% ethanol and 100% acetonitrile before elution in digest buffer . Lysis was followed by a 15-minute centrifugation to pellet the nuclei and insoluble materials. The supernatants were subsequently used as indicated. For HMG-CoA reductase (HMGCR) IP, 2 μg of anti-HMGCR antibody together with 10 μL of a mixture of 1:1 protein A and G coupled sepharose beads  were added to lysates and incubated overnight at 4°C. Proteins from lysate or IP were subjected to SDS-PAGE followed by immunoblotting according to standard procedures  with the samples generated for Western blot analysis using 50 μg total protein per reaction. A recombinant HMGCR provided by the kit was used as a positive control, the recombinant enzyme incubated with 5 μL of the Pravastatin (provided in the kit) was used as negative control, and only lysis buffer was used as blank. Samples, buffers, and substrates were added following the order in the manufacturer's protocol. Absorbance was measured at 340 nm using a microplate reader. The activity is expressed as AU/mg protein where 1 unit (AU) is the amount of HMGCR oxidating 1 μmol of NADPH in a minute at 37°C.t test was applied. For data not meeting the criteria for normality, the Mann–Whitney or Wilcoxon signed-rank test was applied. For the analysis of more than two groups, one-way ANOVA was applied in case of normally distributed data. Correction for multiple comparisons was performed by Dunnett test . Nonparametric data were analyzed using the Kruskal–Wallis test followed by Dunn test  to correct for multiple comparisons. Grouped analyses were tested using two-way ANOVA. To correct for multiple comparisons, Dunnett (comparing groups with respective control group inside of one row), Sidak (comparing groups with respective control group inside of one column or comparing two groups in one row), or Tukey  test was applied. If data were analyzed compared with a hypothetical value of 1 or 100, we used the one-sample t test for normally distributed data and the one-sample Wilcoxon test when nonparametric testing was suggested. log-rank test (Mantel–Cox) was used to calculate significance of differences between survival curves. Statistical analysis was performed using GraphPad Prism . Applied analyses and statistical significances are indicated in the corresponding figures and figure legends. All data are represented as means ± SEM. Differences with P ≤ 0.05 were considered statistically significant. ns, nonsignificant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. All data values and the corresponding statistical tests of each graph are available.All data were tested for normality applying the D'Agostino and Pearson or Shapiro–Wilk test. For the comparison of two groups, an unpaired two-tailed Student The Cancer Genome Atlas (TCGA) RNA sequencing (RNA-seq) data were downloaded from TCGAbiolinks R/Bioconductor package. Only primary tumor samples from TNBC were retained in the analysis. In total, 180 RNA-seq samples with survival data available were analyzed. A list of TCGA barcodes is provided in Supplementary Table S1. Downstream analysis was performed with R (v4.2.2). Read counts were normalized by the library size (count per million). Genes quantified in less than 75% of the dataset were filtered out. Survival analysis, including Cox proportional hazard, was performed with “survival” and “survminer” R package.The proteomic data generated in this study are publicly available via ProteomeXchange with identifier PXD039463. Other data generated in this study are available within the article and its Supplementary Data or from the corresponding author upon reasonable request.+ and Vδ2+ T cells . The expression of the activating natural killer–cell receptor NKG2D, which mediates the cytolytic activity of γδ T cells . It has been reported that the overt expansion of αβ T cells, their genetic modification or their stimulation after transfer to a patient can result in T-cell exhaustion and functional failure . Expanded γδ T cells were used for experiments between day 14 and day 35 after starting the expansion because the cells have reached a stable phenotype at this time window and cytotoxicity remained stable .To test the cytotoxic potential of human γδ T cells toward patient-derived triple-negative BCSCs, we expanded γδ T cells from PBMCs of healthy donors using concanavalin A (ConA) stimulation. As described previously , expansi T cells , was upr failure . Therefoin vitro. Expanded γδ T cells from 3 healthy donors killed all tested patient-derived triple-negative BCSC lines  in an effector to target ratio-dependent manner . Both Vδ1+ and Vδ2+ T cells displayed similar cytotoxicity in response to BCSC1, BCSC3, and BCSC5 . Among those, we found four of them, namely CXCR4, CXCR6, CCR4, and CCR7, to be differentially expressed in αβ, γδ, Vδ1 T cells . The exp T cells . Besides T cells . However+ T cells and less than 10% of Vδ1+ T cells to minimize heterogeneity.Taken together, ConA-expanded γδ T cells most efficiently recognized and killed BCSC5 among the tested BCSCs, and exhibited a robust migration toward BCSC5 CM. Therefore, we focused our studies on BCSC5 and conducted further experiments with γδ T-cell cultures containing mainly Vδ2E240A, and expression was verified by flow cytometry . The median survival of the mice after treatment started increased from 28 days for vehicle-treated animals to 33 and 38 days for γδ T cell-treated or γδ T cell MMP14-treated mice, respectively .After establishing a system to maximize γδ T-cell targeting of BCSC eviously . Mice weectively . Althougectively , right. in vitro cultured BCSC5 . We observed that the proportion of degranulating γδ T cells was significantly reduced from 75% to approximately 30% when compared with the response toward ed BCSC5 , left. Sed BCSC5 , right. ed BCSC5 . We alsoed BCSC5 . γδ T-cein vivo, we combined the adoptive transfer of γδ T cells with biweekly applications of a clinically relevant anti–PD-1 antibody (nivolumab). However, the combination treatment did not improve the overall survival of the xenograft-bearing mice but showed a slight reduction in the tumor growth kinetics in vivo  signature. However, proteins usually expressed by MSCs were significantly downregulated in xenograft-derived tumor cells and, vice versa, proteins usually lowly expressed in MSCs were upregulated . These f subtype .in vivo drastically changed the expression levels of well-known γδ T-cell ligands and other molecules involved in cancer cell recognition by γδ T cells (https://www.cancer.gov/tcga) datasets for patients with TNBC scoring for the expression level of Fas, MICA/B, TRAILR1/2, and ICAM-1 individually. Cox proportional hazards analysis failed to reveal significant prolonged survival for patients with high expression of each of the analyzed proteins . This analysis suggests that each of these proteins individually cannot predict suitability for eventual immunotherapies using γδ T cells.In addition to changes affecting stemness, the transfer and growth of BCSC5  T cells . We foun T cells . We vali T cells . All the T cells . Althoug T cells . While T T cells . Taken tNext, we aimed to elucidate the mechanisms involved in the killing of BCSC5 by γδ T cells to narrow down which phenotypic changes might be mainly responsible for the immune escape of xenografted BCSC5. We investigated the role of Fas, MICB, TRAILR1/2, and ICAM-1 in the recognition and subsequent killing of BCSC5 by performing cytotoxicity experiments using blocking antibodies. We found that γδ T cell–mediated killing of BCSC5 involves Fas-FasL interactions, MICA/B engagement and, most drastically, ICAM-1 binding . In contHMGCR. Indeed, SREBP-SCAP protein levels were significantly reduced in xenograft-derived tumor cells . Consequently, HMGCR transcripts were reduced both in the xenograft-derived tumor cells as well as in the original patient tumors indicating that in vivo differentiation of BCSCs reduces HMGCR both in mouse models and in patients . Furthermore, HMGCR activity was significantly reduced in lysates obtained from xenograft-derived tumor cells (+ T cells in the context of butyrophilin 3A1 (BTN3A1) and 2A1  and TNBC patient-derived samples (0.5924). We clustered patients with TCGA TNBC by the expression levels of both BTN2A1 and BTN3A1 and found that high expression of both proteins significantly correlated with increased survival. Patients with high levels of BTN2A1 and BTN3A1 mRNA had a two times lower 10-year mortality risk . CD4+ memory T cells, resting and activated, CD8+ T cells, and antitumor M1 macrophages were enriched in patients with TNBC with high average expression. In contrast, M2 tumor-promoting macrophages were significantly reduced. The assessment of tumor-infiltrating human γδ T cells by deconvolution has proven to be challenging, therefore we analyzed genes specifically upregulated in human γδ T cells that have been identified by machine learning approaches  and their non–stem cell counterparts (CD44hiCD24hi). Sorted stemlike cells from the triple-negative SUM149 cell line and from PDX401 cells were less efficiently killed by γδ T cells compared with non–stem cell counterparts (in vivo. To the best of our knowledge, only one other study investigated the response of γδ T cells against BCSCs and nonstem cells derived from ras-transformed human mammary epithelial cells, and found that both cell populations were equally resistant to γδ T cell–mediated killing (Our observation that BCSCs can be better recognized by γδ T cells than their differentiated progeny apparently opposes previous reports using the expression of CD44 and CD24 to define stemlike cells (CD44terparts . This st killing . Yet, boIn addition, our results highlight a previously unnoticed dichotomy, namely that IFNγ responses and cytotoxicity by γδ T cells do not necessarily correlate. Zoledronate enhanced cytotoxicity by γδ T cells but failed to promote IFNγ secretion. In contrast, IFNα treatment increased γδ T cell–mediated IFNγ secretion but failed to enhance γδ T cell-mediated cytotoxicity. This result suggests that IFNα might represent an attractive tool to be used in combinatorial therapies to induce synergistic effects of γδ and αβ T-cell responses.in vivo growth of these BCSCs leads to their differentiation into cells that lost stemness and ligands to activate γδ T-cell responses and thereby, escaped from efficient killing by γδ T cells. Still, γδ T cells residually killed in vivo differentiated cells by recognizing pAgs. This killing could be increased to the level of BCSC5 killing by zoledronate. In all, we propose that a combinatorial therapy using γδ T cells and zoledronate represents a valuable approach to target triple-negative BCSCs and non–stem cells alike. Furthermore, IFNα treatment could induce IFNγ production by γδ T cells, and thereby induce a first source of IFNγ promoting ICAM-1 expression, T-cell entry into solid tumors (In summary, our data show that patient-derived triple negative BCSCs are targetable by expanded γδ T cells. However, d tumors  and furtSupplementary Video S1 UTSupplementary Video S1 UTClick here for additional data file.Supplementary Video S2 MMP14Supplementary Video S2 MMP14Click here for additional data file.Supplementary Figures and Figure LegendsSupplementary Figures and Figure LegendsClick here for additional data file.Supplementary Table S1Supplementary Table S1Click here for additional data file.


H)-one, a heterocycle of importance as a pharmaceutical building block, are presented.The synthesis and crystal structure of 5-(trifluoromethyl)-1,3,4-thiadiazol-2-1,3,4-thiadiazol-2(3H)-one (5-TMD-2-one), a compound containing the pharmacologically important heterocycle 1,3,4-thiadiazole, is presented. The asymmetric unit comprises six independent molecules (Z′ = 6), all of which are planar. The r.m.s. deviations from each mean plane range from 0.0063 to 0.0381 Å, not including the CF3 fluorine atoms. Within the crystal, two of the molecules form hydrogen-bonded dimers that in turn combine with inversion-related copies to form tetrameric constructs. Similar tetramers, but lacking inversion symmetry, are formed by the remaining four molecules. The tetramers are linked into tape-like motifs by S⋯O and O⋯O close contacts. The environments of each symmetry-independent molecule were compared via a Hirshfeld surface analysis. The most abundant atom–atom contacts are between fluorine atoms, while the strongest result from N—H⋯O hydrogen bonds.The synthesis and crystal structure of C The reported findings provide insights into the molecular properties and biological activities of 1,3,4-thiadiazole derivatives, contributing to the development of novel therapeutic agents. With the importance of 1,3,4-thiadiazoles in drug discovery research in mind, this paper reports the synthesis and crystal structure of 5-(trifluoromethyl)-1,3,4-thiadiazol-2, being 0.065 (8) Å for C3D, the trifluoromethyl carbon of molecule D. The only internal degree of freedom is the torsion of the trifluoromethyl group, which is disordered in all six symmetry-independent molecules in the structure. Indeed, the CF3 orientations and the refined occupancies of the disorder components, which range from 0.500 (5):0.500 (5) for molecule D to 0.908 (2):0.092 (2) for molecule F, are the only significant differences between the six molecules.The molecular structure of 5-TMD-2-one consists of a 1,3,4-thiadiazone ring, essentially a flat pentagonal heterocycle with two adjacent nitrogen atoms, each flanked by carbon atoms, with a sulfur atom completing the ring. The simplicity of the molecular structure notwithstanding, the crystal structure of 5-TMD-2-one is far more complex, as the asymmetric unit contains six molecules  as at 180 K.The crystals were observed to shatter when cooled to 90 K, but remained intact and gave sharp diffraction at 180 K. This observation prompted us to investigate whether warming the crystals might lead to a simpler crystal structure, 3.A and B (with inversion-related copies) and by molecules C, D, E and F. Within the chosen asymmetric unit, molecules A and B are joined by one short N1A—H1A⋯O1B [D–Ad = 2.726 (2) Å] and one longer N1B—H1B⋯O1A [D–Ad = 3.328 (2) Å] hydrogen bond, leading to B—H1B⋯O1Ai  hydrogen bonds, producing A and B molecules are in close contact  contacts, forming tape-like structures parallel to (011) that extend along the [100] direction. For molecules C, D, E and F, the individual motifs are similar . The most abundant contacts in each case are F⋯F (shown in blue and green), ranging from 39.8% in molecule A  to 25.6% in molecule E .Atom–atom contact two-dimensional fingerprint plots calculated using  A Fig. 4a to 25. E Fig. 4e.4.et al., 2016et al., 2012H)-one, LAPSAY -one], and triclinic -one.A search of the Cambridge Structural Database -one  there are 655 entries with Z′ = 6 out of over 1.2 million (∼0.05%), so by this criterion alone, the structure of 5-TMD-2-one is unusual, though not unprecedented.Although crystal structures with rock 2016 estimate5.Synthesis of 2-amino-5-trifluoromethyl-1,3,4-thiadiazole3COOH and 15.0 mL of POCl3 were slowly added over about 30 min. The reaction was maintained for 3 h, during which time, a large amount of HCl gas was produced. After completion of HCl gas liberation, the reaction mixture was poured into 100 mL of cold water with stirring and the pH adjusted to 9 with 50% NaOH solution, to give a solid precipitate. The product, 2-amino-5-trifluoromethyl-1,3,4-thiadiazole, was filtered, washed with cold water and dried at 363 K (20.6 g).To a clean and dry 1 L round-bottom flask, 14.5 g of thiosemicarbazide suspended in 500 ml of 1,4 dioxane was added, with stirring. 12.0 ml of CFSynthesis of 5-TMD-2-one2 were added slowly  while maintaining the temperature at 263–268 K with continued stirring. After 2 h, 100 ml of H2O were added with warming up to 333–353 K and stirred for a further 3 h. The reaction mixture was then cooled to room temperature, 150 mL of CH2Cl2 were added, the organic layer separated and a further 150 ml CH2Cl2 were added. The combined organic layers were washed with water twice and dried with sodium sulfate, then finally distilled completely. The crude product was purified by chromatography over SiO2 . The resulting product, pure 5-TMD-2-one (12.5 g) was recrystallized from hexane. MS m/z: 169.12 (M − H)+.In a 250 ml round-bottomed flask, 20.6 g of 2-amino-5-trifluoromethyl-1,3,4-thiadiazole was suspended with 150 ml conc. hydrochloric acid, with stirring. The reaction mixture was cooled to between 263 and 268 K. Then, 350 mL of aqueous NaNOA generalized reaction scheme is presented in Fig. 56.Uiso parameters set to 1.2Ueq of their attached nitrogen atom. To ensure satisfactory refinement of the disordered CF3 groups, a combination of constraints (EADP in SHELXL) and restraints  were employed.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989023004267/vm2283sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989023004267/vm2283Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023004267/vm2283Isup3.cmlSupporting information file. DOI: 2263179CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Correction to: Pediatric Radiology (2022) 52:1711–1718https://doi.org/10.1007/s00247-022-05416-5“Diagnostic Radiology Residents” Men – 3,459 (73.5)“Diagnostic Radiology Residents” Women – 1,244 (26.5)“Diagnostic Radiology Residents” Total Non-duplicated – 4,703“U.S. Medical Graduates” Total Non-duplicated – 20,390“Diagnostic Radiology Applicants” Total Non-duplicated – 2,405A corrected version of Table Men – 3,271 (72.2)Women – 1,260 (27.8)Total Non-duplicated – 4,531Of note, a comparison of this 2020 data with the 2010 data published by Chapman et al. [5] shows that compared to the 2010 demographic data (below) there was no significant change in the number or percentage of men and women in DR and IR residency training programs:On page 1714 within the paragraph beginning with “By the 2020 U.S. Census. Women…”, the correct percentage for the women who were diagnostic radiology residents is 26.5% (instead of 35.9%). The point being made about underrepresentation remains valid.


Ogr1 and Tdag8 deficiency was assessed in spontaneous and acute murine colitis models. Disease severity was assessed using clinical scores. Colon samples were analyzed using quantitative polymerase chain reaction (qPCR) and flow cytometry (FACS). In acute colitis, Ogr1-deficient mice showed significantly decreased clinical scores compared with wildtype (WT) mice, while Tdag8-deficient mice and double knockout (KO) mice presented similar scores to WT. In Il-10-spontaneous colitis, Ogr1-deficient mice presented significantly decreased, and Tdag8-deficient mice had increased inflammation. In the Il10−/− × Ogr1−/− × Tdag8−/− triple KO mice, inflammation was significantly decreased compared with Tdag8−/−. Absence of Ogr1 reduced pro-inflammatory cytokines in Tdag8-deficient mice. Tdag8−/− had significantly more IFNγ+ T-lymphocytes and IL-23 T-helper cells in the colon compared with WT. The absence of OGR1 significantly alleviates the intestinal damage mediated by the lack of functional TDAG8. Both OGR1 and TDAG8 represent potential new targets for therapeutic intervention. G-protein-coupled receptors (GPRs), including pro-inflammatory ovarian cancer GPR1 (OGR1/GPR68) and anti-inflammatory T cell death-associated gene 8 (TDAG8/GPR65), are involved in pH sensing and linked to inflammatory bowel disease (IBD). OGR1 and TDAG8 show opposite effects. To determine which effect is predominant or physiologically more relevant, we deleted both receptors in models of intestinal inflammation. Combined  OGR1 and TDAG8 are pH-sensing GPRs that belong to the GPR4 subfamily. They are activated by acidic extracellular pH ,3, reachTdag8 deficiency exacerbates intestinal inflammation and fibrosis in the dextran sodium sulfate (DSS)-induced chronic colitis  and Tdag8−/− [p < 0.0024]). The histological score of Ogr1-deficient mice showed no significant differences when compared with that of the WT H2O control mice . Lymphocyte infiltration and epithelial damage, used to calculate the histology score, were also assessed separately (Ogr1−/− mice compared with WT mice (p = 0.0028) and Tdag8−/− mice (p < 0.001) upon DSS. In contrast, lymphocyte infiltration remained unchanged in Tdag8−/− mice compared with WT mice upon DSS (p = 0.7228). In the Ogr1−/− × Tdag8−/− double KO mice, lymphocyte infiltration was significantly decreased compared with Tdag8−/− mice (p = 0.0349), indicating that the absence of Ogr1 can also improve inflammation in the context of a Tdag8 deficiency. The epithelial damage score was significantly decreased in Ogr1−/− mice compared with WT (p < 0.001), Tdag8−/− (p = 0.0091), or Ogr1−/− × Tdag8−/− mice (p = 0.0117) upon DSS. In contrast to the lymphocyte infiltration sub-analysis, the epithelial damage score showed no amelioration for the Ogr1−/− × Tdag8−/− double KO mice compared with Tdag8−/− mice or WT mice upon DSS.Histology samples were used to assess the severity of inflammation in colon specimens from all groups using HE staining A. The hirol mice B. The Ogparately . The lym−/−Il-10 spontaneous colitis mouse model was applied. For that, −/−Il-10 × −/−Ogr1 (n = 13), −/−Il-10 × Tdag8−/− (n = 8), and Il-10−/− × Ogr1−/− × Tdag8−/− (n = 9) mice were investigated. As a control, littermates from the respective KO with Il-10-deficiency were used . Successful induction of colitis was confirmed by an overall body weight loss . Loss of OGR1 was protective: −/−Il-10 × −/−Ogr1 mice showed a significantly higher body weight at euthanasia compared with Il-10−/− (p = 0.0275), −/−Il-10 × Tdag8−/− (p = 0.0003), and Il-10−/− × Ogr1−/− × Tdag8−/− mice (p = 0.0339) (Il-10−/− × Ogr1−/− group compared with −/−Il-10 mice (p = 0.0002) or −/−Il-10 × Tdag8−/− mice (p < 0.0001). Before euthanasia, an endoscopy was performed on all mice . In order to extend our studies, the ght loss A. Of not 0.0339) A. The di 0.0339) B and shoall mice C. The ren weight E compare−/−Il-10 × −/−Ogr1 mice showed a significantly lower spleen weight compared with −/−Il-10 animals (p = 0.0057). There was a reduction in spleen weight when −/−Il-10 × Tdag8−/− mice were compared with Il-10−/− × Ogr1−/− × Tdag8−/− mice (p = 0.0221). Il-10−/− × Ogr1−/− mice showed a significantly greater colon length when compared with Il-10−/−  or Il-10−/− × Tdag8−/− mice (p < 0.0112). On the other hand, −/−Il-10 × −/−Ogr1 mice compared with −/−Il-10 mice (p < 0.0001), confirming the protective nature of an Ogr1 deficiency. As expected, −/−Il-10 × Tdag8−/− mice showed a significant exacerbation of the disease compared with −/−Il-10 mice (p < 0.0001), including cryptitis (−/−Il-10 × −/−Ogr1 mice (p = 0.1601) and −/−Il-10 mice (p = 0.2666), but a significantly lower score compared with −/−Il-10 × Tdag8−/− mice (p < 0.0001). These results demonstrate that the protective effect of the Ogr1 depletion is maintained in a Tdag8−/− background. −/−Il-10 × Tdag8−/− mice showed a significantly higher histological score compared with all other groups (p < 0.0001).To further assess the severity of inflammation, the distal 1 cm of the colon from all the specimens were subjected to histology. The histological score of the colon  was signryptitis . Interes−/−Il-10 × −/−Ogr1 mice compared with −/−Il-10 (p = 0.0032), −/−Il-10 × Tdag8−/− (p < 0.001), and Il-10−/− × Ogr1−/− × Tdag8−/− (p = 0.0059) mice.Additionally, a histological score of the small bowel was performed . The resTnf and Il-6 in Il-10−/− × Ogr1−/− mice when compared with Il-10−/− mice  and a significant reduction in Il-6 expression levels (p = 0.0061), suggesting a decreased inflammation in the triple KO mice.Transcriptional analysis showed a trend towards a decreased expression of −/− mice A,B. In cIl-10−/− and Il-10−/− × Tdag8−/− mice compared with Il-10−/− × Ogr1−/− and Il-10−/− × Ogr1−/− × Tdag8−/− animals (+ CD4+ T-cells (Ogr1-deficient mice compared with Il-10−/− mice (p = 0.0085), whereas the number of these cells was unaffected in the absence of Tdag8 (Il-10−/− × Ogr1−/− × Tdag8−/− mice showed a decreased number of TNF+ CD4+ T-cells compared with Il-10−/− mice (p = 0.0330), confirming the data obtained by qPCR analysis. A similar picture emerged when measuring the number of IFN+ CD4+ T-cells (Il-10−/− × Tdag8−/− mice showed a similar number of IFN+ CD4+ T-cells to Il-10−/− mice and a trend toward an increased number compared with Il-10−/− × Ogr1−/− (p = 0.0726) and Il-10−/− × Ogr1−/− × Tdag8−/− mice  reduced this level back to the corresponding control .Histological analysis revealed an increased influx of lymphocytes in the colon of  animals A,B. Subs T-cells  were sigof Tdag8 A. Il-10− T-cells B. Il-10−he colon . This pohe colon . These che colon . The numIl10−/− × Tdag8−/− mice in the colon, we analyzed this cell population in spleen samples by FACS  the pro-inflammatory effects of TDAG8 deficiency became clearly visible and highly significant. We can therefore state that TDAG8 deficiency exacerbates colonic inflammation already triggered by a lack of IL10. Exacerbation was particularly prominent in overall disease scores and when measuring IL6 and F4/80duce IL6  and thusduce IL6 ,42,43. Id before , and in Il-17a was increased in Tdag8-deficient mice in a model of myocardial infarction , version 9.5.1. (733), with analysis of variance [ANOVA]. Normal distribution was analyzed using the Shapiro–Wilk test, followed by Tukey post hoc test A,B or no