{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Tabular":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/20230314_103954_Coloncancer_Vienna_Report.tsv"],"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/checksum.txt"],"Raw":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_08_Tumor-RFP553.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_30_Colon-RFP580.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_24_Tumor-RFP560.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_20_Tumor-RFP550.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_22_Tumor-RFP552.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_34_Colon-RFP579.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_40_Colon-RFP583.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_32_Colon-RFP578.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_42_Colon-RFP582.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_06_Tumor-RFP554.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_04_Tumor-RFP561.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_10_Tumor-RFP551.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_18_Tumor-RFP557.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_16_Tumor-RFP555.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_12_Tumor-RFP556.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_14_Tumor-RFP558.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_36_Colon-RFP584.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_26_Tumor-RFP559.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD058770/OTEX2_230105_19-317_YD_38_Colon-RFP581.raw"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["u.klingmueller@dkfz-heidelberg.de"],"submitter":["Marcel Schilling"],"technology_type":["Mass Spectrometry","Data-independent acquisition"],"software":[""],"submitter_keywords":["Mouse","Scrna-seq","Metastatic colorectal cancer (mcrc)","Egfr signaling"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD058770"],"tissue":["Bone Marrow"],"sample_protocol":["Sorted myeloid cells were washed once with cold PBS and resuspended in RIPA lysis buffer 25mM trizma hydrochloride (Tris-HCL) ph:7.6 (T1503; Sigma Aldrich), 1% gr/ml sodium deoxycholate (D6750; Sigma Aldrich), 150mM sodium chloride NaCl (1.06404; Sigma Aldrich), 0.1% w/v sodium dodecyl sulfate SDS (1610418; Biorad), 1% v/v nonidet NP-40 (N-6507; Sigma Aldrich) supplemented with 1mM sodium orthovanadate (Na3VO4) (S6508; Sigma Aldrich), 10mM sodium fluoride (NaF) (7681-49-4; Thermo Fischer Scientific), 1ug/ml Aprotinin (A1153-1MG; Sigma Aldrich), 0.1mg/ml 4-2-(Aminoethyl) benzenesulfonylfluoride (AEBSF) (1248390500; MERCK), 250U Benzonase (70746-4; Sigma Aldrich), 10U DNAse I (DN25-1G; Scient Labo) and 1x PhosphoSTOP (4906845001; Sigma Aldrich). Samples were incubated for 30min at 4°C and frozen in liquid nitrogen. Protein concentrations were determined by employing the Pierce BCA Protein Assay Kit (23227; Thermo Fisher). In total, 2µg of protein per sample was used for further processing. The disulfide bonds of proteins were reduced with 40mM Tris(2-carboxyethyl) phosphine (TCEP) and then alkylated with 10mM chloroacetamide (CAA) for 60min at 37°C. Protein digestion and clean-up were performed using an adapted version of the automated paramagnetic bead-based single-pot, solid-phase-enhanced sample-preparation (Auto-SP3) protocol (Müller et al., 2020) on the Bravo liquid handling platform (Agilent). Briefly, for bead preparation, Sera-Mag Speed Beads A and B (Cytiva) were vortexed until the pellet was dissolved. The suspension was placed on a magnetic rack, and after one minute, the supernatant was removed. The beads were taken off the magnetic rack and suspended in water. This procedure was repeated three times. A total of 10µl of bead A was combined with 10µl of bead B, and the final volume was corrected to 100µl with H2O. To each sample, a total of 5µl of A+B beads were added. To induce the binding of the proteins to the beads, ethanol was added to each sample to a final 50% concentration (v/v). Samples were then incubated for 15min at room temperature and 800rpm. After the incubation step, samples were placed again on a magnetic rack, and after one minute, the supernatant was removed. Samples were taken off the magnetic rack and suspended in 80% ethanol. This procedure was repeated three times. Finally, samples were reconstituted in 100mM TEAB buffer containing trypsin (enzyme/protein ratio of 1:25) and digested overnight on a shaker at 37°C and 1000rpm. After digestion, the recovered peptides were dried by vacuum centrifugation (1300rpm. at 45°C) and stored at -80°C until use. Nano-flow LC-MS/MS was performed by coupling an Ultimate 3000 HPLC (Thermo Fisher Scientific, USA) to an Orbitrap Exploris mass spectrometer (Thermo Fisher Scientific, Germany). Peptide samples were dissolved in 15µl loading buffer (0.1% formic acid (FA), 2% ACN in MS-compatible H2O), and 2µl were injected for each analysis. The samples were loaded onto a pre-column (trap) at higher flowrates (PEPMAP 100 C18 5µm 0.3mmx5mm, Thermo Scientific), using a loading pump and then a valve was switched to delivered to an analytical column (100µm × 30cm, packed in-house with Reprosil-Pur 120 C18-AQ, 1.9µm resin, Dr. Maisch) at a flow rate of 3µl/min in 98% buffer A (0.1% FA in MS-compatible H2O). After loading, peptides were separated using a 120min gradient from 2% to 38% of buffer B (0.1% FA, 80% ACN in MS-compatible H2O) at 350nL/min flow rate. The Orbitrap Exploris 480 was operated in data-independent (DIA) mode, with a m/z range of 350–1400. Full scan spectra were acquired in the Orbitrap at 120’000 resolution after accumulation to the set target value of 300% (100% = 1e6) and maximum injection time of 45ms. The full scans were followed by DIA scans. A total of 47 isolation windows were defined, with a m/z range of 406-986. DIA scan spectra were acquired at 30’000 resolution after accumulation to the set target value of 1000% (100%=1e5) and maximum injection time of 54ms. Normalized collision energy (NCE) was set to 28%."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["All DIA–MS data files were analyzed with a directDIA workflow using Spectronaut 17.1 (Biognosys, Zurich, Switzerland). For the Pulsar search, the UniProt mouse-reviewed canonical reference proteome (20,404 entries, downloaded on December 7th, 2022). The default settings for database match include full specificity trypsin digestion, peptide length of between 7 and 52 amino acids, and maximum missed cleavage of 2. N-terminal methionine was removed during preprocessing of the protein database. Carbamidomethylation at cysteine was used as a fixed modification, and protein N-terminal acetylation and methionine oxidation were set as variable modifications. The false discovery rates (FDRs) were set as 0.01 for the peptide-spectrum match (PSM), peptide and protein identification. Other Spectronaut parameters for identification included maximum intensity as MZ extraction strategy, 0.01 precursor Qvalue Cutoff, 0.2 Precursor PEP Cutoff, stripped sequence as single hit definition. For quantification, identified (Qvalue) was set for precursor filtering, MaxLFQ as protein LFQ method and MS2 quantification with area as quantity type. For further analysis, only those proteins without missing values were considered. For the analysis of the proteome data the public server at usegalaxy.org was used (Afgan et al., 2016). Analysis was based on the R/Bioconductor package limma (v 3.5). Statistical testing of significance was performed using the voom() function (Law et al., 2014)."],"omics_type":["Proteomics"],"labhead":["Prof. Ursula Klingmüller"],"instrument_platform":[""],"labhead_affiliation":["German Cancer Research Center (DKFZ),Division Systems Biology of Signal Transduction, INF 280, 69120 Heidelberg"],"submission_type":["PARTIAL"],"species":["Mus Musculus (mouse)"],"submitter_mail":["m.schilling@dkfz.de"],"publication":["42230982 Fari O, Neuhofer A, Holcmann M, Chontorotzea T, Barozzi I, Mohr T, Blauensteiner B, Krauß D, Xiao YT, Lang M, Frey DL, Helm B, Klingmüller U, Farlik M, Sibilia M. EGFR deletion in myeloid cells reprograms the immunosuppressive landscape of colorectal cancer. Cell Death Differ. 2026 10.1038/s41418-026-01774-x"],"submitter_affiliation":["Division Systems Biology of Signal Transduction, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg"],"submitter_country":["Germany"],"pubmed_abstract":["Despite advances in the treatment of metastatic colorectal cancer (mCRC), it remains the second leading cause of cancer-related mortality, with limited effective therapeutic options. While EGFR inhibition is a standard first-line therapy for mCRC patients lacking KRAS mutations, resistance frequently develops, limiting its clinical benefit. Murine CRC models have shown that EGFR deletion in myeloid cells reduces tumor burden, and the presence of EGFR-positive myeloid cells is associated with poor prognosis in mCRC patients. However, the role of these cells in the therapeutic response to anti-EGFR therapies remains poorly understood. In this study, we integrated mouse models, single-cell RNA sequencing (scRNAseq), proteomics, and patient-derived mCRC datasets to investigate how EGFR signaling in myeloid cells shapes the tumor microenvironment (TME). In a preclinical therapeutic trial, we demonstrate that EGFR deletion in myeloid cells of tumor-bearing CRC mice reduced tumor growth, whereas EGFR loss in intestinal epithelial tumor cells alone had no therapeutic impact. EGFR deletion also decreased the abundance of F4/80<sup>hi</sup> macrophages, particularly the Spp1<sup>+</sup> and C1qc<sup>+</sup> subsets, and reduced inflammatory pathways like TGFβ, IFNγ, and JAK/STAT signaling in myeloid cells. These changes altered myeloid-T cell interactions, resulting in a less immunosuppressive TME characterized by reduced immune checkpoint expression. Furthermore, we identified thrombospondin-1 (THBS1) as a myeloid-derived ligand interacting with T cells, and confirmed its regulation by EGFR signaling through proteomic analysis. Analysis of human CRC datasets revealed that high EGFR and THBS1 expression correlates with poor patient outcomes. Collectively, these findings establish EGFR signaling in myeloid cells as a critical regulator of the immunosuppressive TME and suggest that targeting EGFR within specific myeloid subsets could represent a promising therapeutic strategy in mCRC."],"pubmed_title":["EGFR deletion in myeloid cells reprograms the immunosuppressive landscape of colorectal cancer."],"pubmed_authors":["Fari Ourania O, Neuhofer Angelika A, Holcmann Martin M, Chontorotzea Tatiana T, Barozzi Iros I, Mohr Thomas T, Blauensteiner Bernadette B, Krauß Dana D, Xiao Yu-Tian YT, Lang Michaela M, Frey Dario L DL, Helm Barbara B, Klingmüller Ursula U, Farlik Matthias M, Sibilia Maria M"],"additional_accession":[]},"is_claimable":false,"name":"Deletion of Egfr in myeloid cells counteracts an immuno-suppressive tumor microenvironment in CRC","description":"Despite advances in metastatic colorectal cancer (mCRC) treatment, it remains the second-leading cause of cancer-related deaths, with limited therapeutic options. EGFR inhibition is a first-line treatment for mCRC without KRAS mutations, but resistance persists in some patients. Preclinical murine CRC models have shown that EGFR deletion in myeloid cells results in smaller tumors, while the presence of EGFR-positive myeloid cells correlate with poor survival in mCRC patients. However, the role of these cells in tumor progression remains unclear. In this study, we used preclinical mouse models, scRNA-seq, proteomics, and mCRC patient data to investigate the impact of EGFR expression in myeloid cells on the tumor microenvironment (TME) and explore its therapeutic potential. We demonstrate that EGFR deletion in myeloid cells of tumor-bearing CRC mice reduces tumor growth, while EGFR deletion in tumor cells alone has no therapeutic effect. EGFR disruption also decreases F4/80hi macrophages, particularly Spp1+ and C1qc+ subsets, and reduces inflammatory signaling like TGFβ, IFNγ, JAK/STAT in myeloid cells.  This, in turn, affects their interaction with T cells, promoting a shift towards a less immunosuppressive TME. Additionally, we identify reduced immune checkpoint expression and thrombospondin-1 (Thbs1) as a myeloid-derived ligand interacting with T-cells. Validation in human CRC shows that high expression of EGFR and THBS1 correlates with poorer prognosis. These findings highlight EGFR signaling in myeloid cells as a key modulator of the TME and suggest targeting EGFR in specific myeloid subsets could improve CRC treatment strategies.","dates":{"publication":"2026-06-08","submission":"2024-12-11"},"accession":"PXD058770","cross_references":{"TAXONOMY":["NEWT:108018","NEWT:377960","NEWT:1131","NEWT:190486","NEWT:2737160","NEWT:2042546","NEWT:148942","NEWT:193528","NEWT:295546","NEWT:258594","NEWT:1126","NEWT:1129","NEWT:89187","NEWT:309807","NEWT:89184","NEWT:309800","NEWT:57577","NEWT:156563","NEWT:1323429","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:44270","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:93217","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:10034","NEWT:10031","NEWT:82569","NEWT:661410","NEWT:488003","NEWT:224326","NEWT:109376","NEWT:376619","NEWT:3765","NEWT:2433","NEWT:1190602","NEWT:3767","NEWT:433727","NEWT:135622","NEWT:672127","NEWT:656061","NEWT:37965","NEWT:656064","NEWT:3760","NEWT:36630","NEWT:381198","NEWT:1176302","NEWT:418106","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:44250","NEWT:77151","NEWT:317447","NEWT:698761","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:1177","NEWT:11308","NEWT:12637","NEWT:2164133","NEWT:1180","NEWT:2767846","NEWT:1343074","NEWT:314344","NEWT:108061","NEWT:143292","NEWT:2767842","NEWT:314345","NEWT:224308","NEWT:3134131","NEWT:49928","NEWT:160621","NEWT:110368","NCBITaxon:5204","NEWT:1916073","NEWT:1303873","NEWT:305400","NEWT:1310161","NEWT:1230383","NEWT:77133","NEWT:160620","NCBITaxon:77643","NEWT:1310165","NEWT:114727","NEWT:5516","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:32224","NEWT:11320","NEWT:1410384","NEWT:1310173","NEWT:1072569","NEWT:1072568","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:135666","NEWT:419481","NEWT:5507","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:1143","NEWT:1336794","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:709964","NEWT:3710","NEWT:393133","NEWT:65125","NEWT:3712","NEWT:3711","NEWT:3714","NEWT:65121","NEWT:3716","NEWT:3715","NEWT:2065263","NEWT:1549675","NEWT:177437","NEWT:9014","NEWT:10891","NEWT:118221","NEWT:32201","NEWT:34869","NEWT:226126","NEWT:1616117","NEWT:34865","NEWT:28174","NEWT:550537","NEWT:284812","NEWT:284811","NEWT:2902907","NEWT:3701","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:1246791","NEWT:34875","NEWT:10882","NEWT:1431297","NEWT:9005","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:1883426","NEWT:657321","NEWT:1423687","NEWT:657322","NEWT:10886","NEWT:3707","NEWT:3708","NEWT:536231","NEWT:219813","NEWT:1301262","NEWT:319224","NEWT:2305479","NEWT:215451","NEWT:460519","NEWT:292415","NEWT:272843","NEWT:272844","NEWT:1348799","NEWT:55784","NEWT:485","NEWT:223074","NEWT:2499525","NEWT:467144","NEWT:142786","NEWT:28196","NEWT:2853422","NEWT:487","NEWT:1679718","NEWT:387344","NEWT:657318","NEWT:480","NEWT:67767","NEWT:46835","NEWT:216778","NEWT:376686","NEWT:237610","EFO:0001352","NEWT:219809","NEWT:219808","NEWT:119562","NEWT:367737","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:141469","NEWT:272831","NEWT:987059","NEWT:491","NEWT:422203","NEWT:460523","NEWT:2422","NEWT:2423","NEWT:3755","NEWT:85694","NEWT:85692","NEWT:911045","NEWT:3750","NEWT:1195466","NEWT:2483192","NEWT:85698","NEWT:291146","NEWT:233271","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:278161","NEWT:415978","NEWT:410289","NEWT:191701","NEWT:9054","NEWT:472","NEWT:29459","NEWT:34839","NEWT:476","NEWT:1380096","NEWT:28128","NEWT:470","NEWT:41122","NEWT:1328300","NEWT:748693","NEWT:106299","NCBITaxon:50557","NEWT:63363","NEWT:3735","NEWT:1002697","NEWT:29491","NEWT:911022","NEWT:446","NEWT:153481","NEWT:714485","NEWT:445","NEWT:427924","NEWT:2003758","NEWT:29486","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:3726","NEWT:1120755","NEWT:1862567","NEWT:591090","NEWT:30455","NEWT:28141","NEWT:9030","NEWT:9031","NEWT:3728","NEWT:29471","NEWT:108931","NEWT:219799","NEWT:1325292","NEWT:219798","NEWT:548","NEWT:150475","NEWT:546","NEWT:1431340","NEWT:256737","NEWT:51350","NEWT:8694","NEWT:8692","NEWT:542","NEWT:394503","NEWT:13076","NEWT:219785","NEWT:451515","NEWT:8673","NEWT:550","NEWT:10804","NEWT:52694","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:40686","NEWT:1225786","NEWT:366497","NEWT:771870","NEWT:8665","NEWT:51338","NEWT:771875","NEWT:377142","NEWT:9995","NEWT:28112","NEWT:49012","NEWT:28110","NEWT:10832","NEWT:311339","NEWT:507601","NEWT:520","NEWT:64657","NEWT:186441","NEWT:2585209","NEWT:643680","NEWT:214092","NEWT:40697","NEWT:13095","NEWT:162425","NEWT:242124","NEWT:9986","NEWT:8654","NEWT:536","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:80833","NEWT:479096","NEWT:28108","NEWT:28104","NEWT:1114957","NEWT:568703","NEWT:568708","NEWT:480020","NEWT:51315","NEWT:52641","NEWT:353152","NEWT:116597","NEWT:353154","NEWT:353153","NEWT:116598","NEWT:227377","NEWT:1774284","NEWT:27689","NEWT:207769","NEWT:40674","NEWT:51329","NCBITaxon:1763","NEWT:443906","NEWT:6055","NEWT:13037","NEWT:518","NEWT:519","NEWT:941638","NEWT:51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