Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD067046/xb13494.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD067046/xb13493.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD067046/xb13491.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD067046/xb13490.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD067046/647063_241105_ALS_xb13493_phospho_MF.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD067046/647064_241105_ALS_xb13491_phospho_MF.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD067046/647062_241105_ALS_xb13494_phospho_MF.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD067046/647065_241105_ALS_xb13490_phospho_MF.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD067046/241105_ALS_xb13491_phospho_MF.mzXMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD067046/241105_ALS_xb13493_phospho_MF.mzXMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD067046/241105_ALS_xb13494_phospho_MF.mzXMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD067046/241105_ALS_xb13490_phospho_MF.mzXMLprimaryOK200amanda_smythers@dfci.harvard.eduAmanda SmythersMass SpectrometryBottom-up proteomicsNot availablePhosphoproteomicsPancreatic cancerTmthttps://www.ebi.ac.uk/pride/archive/projects/PXD067046Cell Culture2,500,000 C7-TA-PSC cells were plated into 10cm dishes for each respective condition and incubated overnight. 1 hour before treatment with ALP, media was refreshed on the cells to remove any constitutive signals that had accumulated after cell seeding while allowing cells to equilibrate from the media change. 1 hour later, media was refreshed once again with 20μM ALP and cell pellets were collected via trypsinization and frozen at each consecutive timepoint. For phospho-enrichment, cell pellets were lysed with 8 M urea dissolved in 150 uL lysis buffer (50 mM 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS) (pH 8.5), 1% SDS, 1 PhosSTOP (Roche), 1 cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Tablet (Roche)) before clarifying with a 10 min centrifugation at 4 C and 16,000 x g. Supernatant was quantified using the CB-X protein assay (G-Biosciences). All samples were diluted to 1 mg/mL with water, reduced with 5 mM DTT for 30 min, and alkylated with 15 mM iodoacetamide in the dark for 30 min. Samples were desalted using the SP3 method (65), with a 1:1 mixture of two magnetic beads (Cytiva 45152105050250 and 65152105050250). After mixing a 1:10 mixture of beads and protein, protein was precipitated through the addition of 2x volume 100% ethanol for 15 min, before washing with 80% ethanol twice. The beads were then incubated in 75 uL of 50 mM EPPS buffer with 1 μg MS Trypsin (Thermo Fisher) overnight at 37 C, shaking at 800 RPM. Peptides were labeled by adding anhydrous acetonitrile (Sigma) to 30% v/v, followed by TMTpro (Thermo A44520) reagent. 1% of each labeled sample was combined and analyzed unfractionated to ensure labeling efficiency was >97%, and all channels were combined. The combined sample was desalted with a 100 mg C18 Sep-Pac (Waters) following manufacturer’s instructions, before drying with a vacuum centrifuge. Peptides were reconstituted and enriched for phosphopeptides using an Fe-NTA spin column (Thermo A32992). The flowthrough was enriched for further phosphopeptides using a TiO2 spin column (Thermo A32993), both following manufacturer’s instructions. Samples were desalted using C18 StageTips for LC-MS/MS analysis.PrideIn-house software tools were used for database searching and reporter ion quantitation, as previously reported. MS/MS spectra were assigned using the Sequest algorithm and matched to a Uniprot human database (compiled Oct. 2024), with both forward and reverse sequenced combined to enable target-decoy strategy. Database searches were performed with static modifications of cysteine alkylation and TMT on the peptide N-termini and lysine residues. Variable modifications included oxidation of methionine and phosphorylation of serine, threonine, and tyrosine. Neutral phosphate loss was searched and identified as well. The precursor ion tolerance was 50 ppm and the fragment ion tolerance was 0.8 Da (for HCD). Sequest matches were filtered using linear discriminant analysis as previously reported first to a dataset level error of 1% at the peptide level based on matches to reversed sequences. Peptide quantification TMT reporter ions were quantified as previously published. Briefly, TMT signals were isolated with a filter of 0.004 Da and corrected for isotope impurities according to the manufacturer’s instructions. Peptides were only considered quantifiable if the total signal-to-noise for all channels was >160. Statistical analyses of phosphopeptide site localization and quantitation Phosphorylation sites were assigned to specific residues using a modified version of the Ascore algorithm. Phosphorylation sites with Ascore values > 13 (p ≤ 0.05) were considered confidently localized to a particular residue. Student’s t-test was used to assign confidence to changes in phosphopeptide abundance.ProteomicsAmanda SmythersDana-Farber Cancer InstituteCOMPLETEHomo Sapiens (human)amanda_smythers@dfci.harvard.edu41996628 Reavis HD, Chaubey AH, Smythers AL, Tisdale AA, Tracz A, Dimeck AN, Maraszek KE, Cortes Gomez E, Paulo JA, Dasgupta S, Gygi SP, Feigin ME. Alprazolam Reduces Inflammatory Cytokine Production in Pancreatic Cancer-Associated Fibroblasts. Cancer Res Commun. 2026 6(5):1048-1060 10.1158/2767-9764.crc-25-0472Dana Farber Cancer Institute, Harvard Medical SchoolUnited States10.6019/PXD067046Pancreatic ductal adenocarcinoma (PDAC) diagnoses are often accompanied by a number of physical and psychologic symptoms, including anxiety and depression. As a result, many patients are prescribed anxiolytics such as benzodiazepines (BZD) that have unintended effects on the tumor. Previous work from our lab has highlighted that structural differences between BZD compounds may be responsible for different clinical outcomes influenced by their effects on cancer-associated fibroblasts (CAF) within the PDAC tumor microenvironment (TME). In this study, we demonstrate that the commonly prescribed N-substituted triazolobenzodiazepine alprazolam (ALP) abrogates the production of proinflammatory cytokines, including CCL2, CXCL12, interleukin 6 (IL6), and IL8, in human CAFs. This phenotype is unique only to azole-containing BZDs, including midazolam. The ability of ALP to regulate proinflammatory cytokine production is maintained in vivo as ALP-treated mice bearing pancreatic tumors exhibited reductions in IL6 within the tumor interstitial fluid. Mechanistically, an unbiased phosphoproteomic approach revealed that ALP abrogates Toll-like receptor 4-mediated cytokine production in CAFs. These findings cumulatively support that ALP dampens CAF-mediated inflammatory signaling within the PDAC TME.<h4>Significance</h4>The data in the present study focus on the characterization of the effects of ALP on CAF output and provide a compelling basis to suggest that ALP may have a broad impact on remodeling the pancreatic cancer immune landscape and therapeutic response.Alprazolam Reduces Inflammatory Cytokine Production in Pancreatic Cancer-Associated Fibroblasts.Reavis Hunter D HD, Chaubey Aditi H AH, Smythers Amanda L AL, Tisdale Arwen A AA, Tracz Amanda A, Dimeck Alphonse N AN, Maraszek Kathryn E KE, Cortes Gomez Eduardo E, Paulo Joao A JA, Dasgupta Subhamoy S, Gygi Steven P SP, Feigin Michael E MEfalseAlprazolam abrogates TLR4 signaling and reduces inflammatory cytokine production in pancreatic cancer-associated fibroblastsPancreatic ductal adenocarcinoma (PDAC) diagnoses are often accompanied by a number of physical and psychological symptoms, including anxiety and depression. As a result, many patients are prescribed anxiolytics such as benzodiazepines that have unintended effects on the tumor. Previous work from our lab has highlighted that structural differences between benzodiazepine compounds may be responsible for different clinical outcomes influenced by their effects on cancer-associated fibroblasts (CAFs) within the PDAC tumor microenvironment (TME). Here, we demonstrate that the commonly prescribed N-substituted triazolobenzodiazepine alprazolam (ALP) abrogates the production of proinflammatory cytokines including CCL2, CXCL12, IL6, and IL8 in human CAFs. This phenotype is unique only to azole-containing benzodiazepines, including midazolam. The ability of ALP to regulate proinflammatory cytokine production is maintained in vivo, as ALP-treated mice bearing pancreatic tumors exhibited reductions in IL6 within the tumor interstitial fluid. Mechanistically, an unbiased phosphoproteomic approach revealed that ALP abrogates TLR4-mediated cytokine production in CAFs. These findings cumulatively support that ALP dampens CAF-mediated inflammatory signaling within the PDAC TME. 2026-06-082025-08-07PXD067046NEWT:330879NEWT:377960NEWT:2042546NEWT:259447NEWT:295546NCBITaxon:2719036NCBITaxon:1280NEWT:112503NEWT:1129NEWT:309807NEWT:59753NEWT:89184NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:193516NEWT:1073882NEWT:475174NEWT:1117NEWT:498257NEWT:10036NEWT:1590NEWT:10034NEWT:661410NEWT:638632NEWT:224326NEWT:376619NCBITaxon:79857NEWT:1096976NEWT:1589NEWT:135622NEWT:35786NEWT:1580NEWT:399784NEWT:96731NEWT:656064NEWT:383379NEWT:418106NEWT:10029NEWT:913645NEWT:641809NEWT:38815NEWT:317447NEWT:4688NEWT:7719NEWT:868565NEWT:135674NEWT:79329NEWT:30069NEWT:12637NEWT:59729NEWT:2164133NEWT:295105NEWT:108061NEWT:60711NEWT:224308NEWT:3347NEWT:160621NEWT:212790NEWT:110368NEWT:1310161NEWT:77133NEWT:145481NEWT:1310165NEWT:29058NCBITaxon:79824NEWT:44688NEWT:44689NEWT:498211NEWT:347256NEWT:5518NEWT:398007NEWT:1527468NEWT:498217NEWT:498216NEWT:11320NEWT:246196NEWT:246197NEWT:145458NEWT:44685NEWT:161934NEWT:1148NEWT:5508NEWT:3329NEWT:419481NEWT:5507NEWT:410661NEWT:1140NCBITaxon:2157NEWT:1143NEWT:1287689NEWT:1094343NEWT:1462472NEWT:1336795NEWT:644042NEWT:1182590NEWT:3712NEWT:3711NEWT:270643NEWT:2065263NEWT:177437NEWT:10418NEWT:118698NEWT:1616117NEWT:118696NEWT:34865NEWT:52283NEWT:284812NEWT:8175NEWT:43330NEWT:1603293NEWT:44664NEWT:43346NEWT:3702NEWT:1245466NEWT:244366NEWT:1246791NEWT:118694NEWT:2850NEWT:118691NEWT:34871NEWT:33548NEWT:52299NEWT:8165NEWT:96794NEWT:3708NEWT:332648NEWT:44670NEWT:536231NEWT:376219NEWT:219813NEWT:1510NEWT:460519NEWT:1515NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:1303443NEWT:9483NEWT:485NEWT:56636NEWT:2853422NEWT:1679718NEWT:480NEWT:67767NEWT:46835NEWT:109757NEWT:582580NEWT:1185650NEWT:216778NEWT:1502NEWT:128017NEWT:376686NEWT:95486NEWT:1883446NEWT:1233435NEWT:109760NEWT:29031NEWT:317047NEWT:460523NEWT:235443NEWT:1757312NEWT:85694NEWT:108458NEWT:5936NEWT:320637NEWT:1078283NEWT:3750NEWT:983964NEWT:11706NEWT:32644NEWT:527796NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:3747NEWT:1116234NEWT:1255228NEWT:410289NEWT:373153NEWT:472NEWT:940825NEWT:529507NEWT:1071661NEWT:470NEWT:5911NCBITaxon:50557NEWT:39251NEWT:29491NEWT:101841NEWT:446NEWT:153481NEWT:2014887NEWT:33952NEWT:445NEWT:153009NEWT:261756NEWT:166313NEWT:63366NEWT:63367NEWT:215402NEWT:1547NEWT:906914NEWT:9031NEWT:27292NEWT:1050722NEWT:108931NEWT:1293497NEWT:1055524NEWT:150475NEWT:267872NEWT:172269NEWT:9534NEWT:5180NEWT:256737NEWT:9541NEWT:8694NEWT:33936NEWT:8692NEWT:2903NEWT:185579NEWT:13076NEWT:1006581NEWT:8673NEWT:33940NEWT:550NEWT:554NEWT:451516NEWT:552NEWT:1325291NEWT:36185NEWT:1054211NEWT:1225786NEWT:51338NEWT:575412NEWT:28112NEWT:6493NEWT:6494NEWT:6491NEWT:507601NEWT:520NEWT:186441NEWT:643680NEWT:214092NCBITaxon:6157NEWT:13095NEWT:162425NEWT:104105NEWT:216257NEWT:9986NEWT:8654NEWT:8658NEWT:1268063NEWT:8657NEWT:9983NEWT:8655NEWT:5147NEWT:28108NEWT:28104NEWT:407821NEWT:568703NCBITaxon:2NEWT:568708NEWT:986NEWT:52641NEWT:28532NEWT:353152NEWT:298176NEWT:40674NEWT:1194669NEWT:51329NEWT:443906NEWT:519NEWT:2510939NEWT:7394NEWT:6063NEWT:1328388NEWT:1548728NEWT:667127NEWT:9557NEWT:377586NEWT:300641NEWT:39655NEWT:9554NEWT:38323NEWT:475932NEWT:256318NEWT:206411NCBITaxon:6191NEWT:229533NEWT:214053NEWT:80863NEWT:90675NEWT:52638NEWT:57075NEWT:1666905NEWT:8697NEWT:8695NEWT:884204NEWT:1123869NEWT:9544NEWT:9545NEWT:979NEWT:7370NEWT:83906NEWT:99488NEWT:1134506NEWT:255470NEWT:38783NEWT:6426NEWT:33090NEWT:9935NEWT:287889NEWT:305959NEWT:13489NEWT:29829NEWT:92867NEWT:92866NCBITaxon:3055NEWT:51750NEWT:202950NEWT:295027NCBITaxon:11320NEWT:632957NEWT:9925NCBITaxon:9606NEWT:90690NEWT:1436183NEWT:300273NEWT:4232NEWT:416348NEWT:11298NEWT:4236NEWT:7742NEWT:196627NEWT:200308NEWT:200302NEWT:870435NEWT:9913NEWT:9915NEWT:105841NEWT:2666255NEWT:999810NCBITaxon:5693NEWT:28995NEWT:1392998NEWT:380394NEWT:226900NEWT:1266738NEWT:231490NEWT:244704NEWT:7725NEWT:9909NEWT:9902NEWT:430615NEWT:72664NEWT:563041NEWT:36599NEWT:41949NEWT:249161NEWT:326423NEWT:452467NEWT:198822NEWT:36111NEWT:326424NEWT:1678078NEWT:749906NEWT:418985NEWT:749907NEWT:150847NEWT:29835NEWT:431947NEWT:69014NEWT:142809NEWT:302409NEWT:130821NEWT:27606NEWT:1519788NEWT:59202NEWT:9975NEWT:8643NEWT:1159899NEWT:502780NEWT:860688NEWT:13443NEWT:35231NEWT:8644NEWT:1125630NEWT:5141NEWT:46360NCBITaxon:3044782NEWT:202914NEWT:160235NEWT:502779NEWT:297246NEWT:8639NEWT:1928434NEWT:8637NEWT:1114970NEWT:2096NEWT:192222NEWT:9963NEWT:1617910NEWT:637379NEWT:2785785NEWT:411901NEWT:1298722NEWT:8626NEWT:186801NEWT:105884NCBITaxon:9615NEWT:8621NEWT:58334NEWT:58331NEWT:8624NEWT:5116NEWT:8623NEWT:5597NCBITaxon:3077NEWT:194439NCBITaxon:12637NEWT:38727NEWT:218496NEWT:8618NEWT:70448NEWT:300251NEWT:9940NEWT:1247190NEWT:118702NEWT:118700NEWT:2126719NEWT:118705NEWT:6945NEWT:2104NEWT:2627626NEWT:6943NEWT:157546NEWT:870726NEWT:91374NEWT:91373NEWT:474186NEWT:54195NEWT:10136NEWT:38942NEWT:4762NEWT:6941NEWT:307972NEWT:2102NEWT:34NEWT:38949NEWT:1299114NEWT:1590841NEWT:1276473NEWT:2282334NEWT:1247NEWT:70667NEWT:869728NEWT:6938NCBITaxon:39054NEWT:1736309NEWT:4751NEWT:194963NEWT:15819NEW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