RNA-seq profiling of macrophage polarization in THP-1 cells co-cultured with GPNMB-knockdown TNBC spheroids
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1128700 | ENA |
REPOSITORIES: ENA
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Project description:Metastasis remains the leading cause of cancer-related mortality, driven by complex interactions within the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) play a pivotal role in metastatic progression, yet molecular diversity and upstream regulators remain poorly defined. Glycoprotein nonmetastatic melanoma protein B (GPNMB), overexpressed in subsets of tumors including triple-negative breast cancer (TNBC), is implicated in epithelial-mesenchymal transition (EMT) and cancer stemness. Recent single-cell RNA-seq studies have identified GPNMB as a marker of immunosuppressive TAMs associated with poor prognosis, but its mechanistic role in TAM polarization in TNBC has remained unclear. Co-culturing monocytic cells with three-dimensional sphere-forming TNBC cells induces their conversion into GPNMB⁺Siglec-9⁺ tumor-associated macrophages (TAMs). Tumor-derived GPNMB promotes monocyte-to-TAM polarization by inducing secondary GPNMB expression in monocytes, establishing a feed-forward amplification loop. Knockdown of GPNMB in TNBC cells significantly inhibits multiple immunosuppressive TAM subtypes, including Siglec-9⁺ TAM and EMT-associated TAM populations, as inferred from scRNA-seq and validated in patient tumors using bulk RNA-seq deconvolution. Distinct sialylation patterns were identified: tumor-derived GPNMB exhibited α2,3-sialylation, whereas macrophage-derived GPNMB exhibited α2,6-sialylation, enabling differential Siglec-9 recognition. Elevated GPNMB and Siglec-9 expression correlated with poor prognosis in TNBC patient cohorts. Importantly, dual inhibition of Siglec-E (murine Siglec-9 ortholog) and PD-1 suppressed IL-6-dependent EMT, reduced tumor stemness, and significantly limited lung metastasis in vivo. The GPNMB–Siglec-9 axis thus represents a critical glyco-immunological checkpoint driving TAM-mediated metastasis, providing a promising therapeutic target in TNBC.
2025-08-15 | GSE270890 | GEO
Project description:The expression of the soluble protein GPNMB (glycoprotein non-metastatic melanoma protein B) induces an aggressive behavior of tumor cells by promoting proliferation and distant spread. The main molecular feature is the over expression of interleukin Il-33. The gene profile of MCA-1-GPNMB and MCA-1-GPNMB-spheroid cell line was assessed against MCA-1-Mock.
2020-07-30 | E-MTAB-8082 | biostudies-arrayexpress
Project description:Metastasis remains the leading cause of cancer-related mortality, driven by complex interactions within the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) play a pivotal role in metastatic progression, yet molecular diversity and upstream regulators remain poorly defined. Glycoprotein nonmetastatic melanoma protein B (GPNMB), overexpressed in subsets of tumors including triple-negative breast cancer (TNBC), is implicated in epithelial-mesenchymal transition (EMT) and cancer stemness. Recent single-cell RNA-seq studies have identified GPNMB as a marker of immunosuppressive TAMs associated with poor prognosis, but its mechanistic role in TAM polarization in TNBC has remained unclear. Co-culturing monocytic cells with three-dimensional sphere-forming TNBC cells induces their conversion into GPNMB⁺Siglec-9⁺ tumor-associated macrophages (TAMs). Tumor-derived GPNMB promotes monocyte-to-TAM polarization by inducing secondary GPNMB expression in monocytes, establishing a feed-forward amplification loop. Knockdown of GPNMB in TNBC cells significantly inhibits multiple immunosuppressive TAM subtypes, including Siglec-9⁺ TAM and EMT-associated TAM populations, as inferred from scRNA-seq and validated in patient tumors using bulk RNA-seq deconvolution. Distinct sialylation patterns were identified: tumor-derived GPNMB exhibited α2,3-sialylation, whereas macrophage-derived GPNMB exhibited α2,6-sialylation, enabling differential Siglec-9 recognition. Elevated GPNMB and Siglec-9 expression correlated with poor prognosis in TNBC patient cohorts. Importantly, dual inhibition of Siglec-E (murine Siglec-9 ortholog) and PD-1 suppressed IL-6-dependent EMT, reduced tumor stemness, and significantly limited lung metastasis in vivo. The GPNMB–Siglec-9 axis thus represents a critical glyco-immunological checkpoint driving TAM-mediated metastasis, providing a promising therapeutic target in TNBC.
2025-08-15 | GSE271964 | GEO
Project description:The experiments were done using an Orbitrap Fusion or an Orbitrap Fusion Lumos mass spectrometer. Multiplexing was achieved using either TMT10 or TMT11 reagents and the SPS-MS3 method. Basic pH reversed-phase chromatography (bRPLC) was used for off-line pre-fractionation; 12 fractions were analyzed for proteome mappings (PMID: 26700037) and 24 fractions plus a phosphotyrosine-enriched sample for phosphoproteome mappings (PMID: 31606085). Phosphoproteome mappings were done using both HCD fragmentation with Orbitrap fragment ion detection and CID fragmentation with ion trap fragment ion detection (PMID: 29487189, PMID: 31606085). If multiple TMT sets were required to analyze all samples from an experiment two bridge channels pooled from all samples was used to connect the data from the individual TMT sets (PMID: 28892078).
Five proteomics experiments are described in the paper:
(i) 1. Quantitative proteomics of fully polarized macrophages after 4 days of treatment with IL4 or IFNgamma/LPS to determine global proteome landscape of fully polarized THP-1-derived M1-type versus M2a-type macrophages:
Proteome mapping on Orbitrap Fusion (one TMT set)
RAW files: Marneros_4DayTreatment_proteome_fraction01 to ...fraction12
8 samples
Labeling: PMA.1 (126), PMA.2 (127n), IL4.1 (127c), IL4.2 (128n), ILJQ1.1 (128c), ILJQ1.2 (129n), IFNgamma.1 (129c), IFNgamma.2 (130n)
(ii) Time-course quantitative proteomics during the first 24 hours after induction of macrophage polarization in THP-1-derived macrophages to identify protein changes associated with M1-type (IFNgamma/LPS-induced) versus M2a-type (IL-4-induced) polarization:
Proteome mapping on Orbitrap Fusion (three TMT sets: set01, set02, set03)
RAW files:
Marneros_timecourse_proteome_TMTset01_fraction01 to ...fraction12
Marneros_timecourse_proteome_TMTset02_fraction01 to ...fraction12
Marneros_timecourse_proteome_TMTset03_fraction01 to ...fraction12
22 samples
Labeling:
set01; bridge 1 (126), PMA 0min (127n), PMA 10min (127c), PMA 30min (128n), PMA 1h (128c), PMA 2h (129n), PMA 4h (129c), PMA 8h (130n), PMA 24h (130c), bridge 2 (131n)
set02; bridge 1 (126), IL4 10min (127n), IL4 30min (127c), IL4 1h (128n), IL4 2h (128c), IL4 4h (129n), IL4 8h (129c), IL4 24h (130n), IFN 10min (130c), bridge 2 (131n)
set03; bridge 1 (126), IFN 30min (127n), IFN 1h (127c), IFN 2h (128n), IFN 4h (128c), IFN 8h (129n), IFN 24h (129c), bridge 2 (131n)
(iii) Time-course quantitative phosphoproteomics during the first 24 hours after induction of macrophage polarization in THP-1-derived macrophages to identify phosphorylation events associated with M1-type (IFNgamma/LPS-induced) versus M2a-type (IL-4-induced) polarization (same time points as in group 2):
Phosphoproteome mapping on Orbitrap Fusion (three TMT sets: set01, set02, set03)
RAW files:
Marneros_timecourse_phospho_TMTset01_fraction01 to ...fraction06, ...fraction08 to ...fraction24, ...pY
Marneros_timecourse_phospho_TMTset02_fraction01 to ...fraction12, ...fraction16 to ...fraction24, ...pY
Marneros_timecourse_phospho_TMTset03_fraction01 to ...fraction24, ...pY
The following RAW files gave no phosphopeptide quantifications (they are no included in the group of provided RAW files): set01_fraction07, set02_fraction13, set02_fraction14, set02_fraction15.
22 samples
Labeling: as in (ii)
(iv) Quantitative proteomics to assess the effects of the B-Raf inhibitor GDC-0879 or of the MEK inhibitor trametinib on IL-4-induced M2-type polarization and IFNgamma/LPS-induced M1-type polarization on THP-1-derived macrophages after 24 hours of treatment:
Proteome mapping on Orbitrap Fusion (one TMT set)
RAW files: Marneros_BRAF_inhibitor_proteine_fraction01 to ...fraction12
11 samples
Labeling: IL4 1 (131c), IL4 2 (127n), IL4+GDC 1 (131n), IL4+GDC 2 (128n), IL4+TRAM 1 (130c), IL4+TRAM 2 (129n), IFNgamma+LPS 1 (130n), IFNgamma+LPS 2 (127c), IFNgamma+LPS+TRAM 1 (126), IFNgamma+LPS+TRAM 2(129c), IFNgamma+LPS+GDC (128c)
(v) Quantitative phosphoproteomics to assess the effects of the B-Raf inhibitor GDC-0879 on IL-4-induced M2-type polarization and IFNgamma/LPS-induced M1-type polarization on THP-1-derived macrophages after 24 hours of treatment:
Phosphoproteome mapping on Orbitrap Fusion Lumos (one TMT set)
RAW files: Marneros_BRAF_inhibitor_phospho_fraction01 to ...fraction24, ...pY
7 samples
Labeling: IL4 1 (131c), IL4 2 (127n), IL4+GDC 1 (130n), IL4+GDC 2 (128n), IFNgamma+LPS 1 (128c), IFNgamma+LPS 2 (127c), IFNgamma+LPS+GDC (130c)
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2016-09-27 | PXD001598 | Pride
Project description:Myocardial infarction occurs when coronary supply of oxygen and nutrients to a part of the heart is interrupted. In contrast to adult humans, adult zebrafish have a unique ability to regenerate their heart after cardiac injury. To identify potential regulators of the regenerative response, we performed transcriptomics profiling of regenerating coronary endothelial cells (cECs) at 7 days post cryoinjury, when revascularization is vigorous, and identified gpnmb to be upregulated. Gpnmb (glycoprotein non-metastatic melanoma protein B) is a transmembrane glycoprotein implicated in inflammation resolution and tissue regeneration. Expression analyses at earlier time points revealed gpnmb specific expression in infiltrating macrophages post cryoinjury in zebrafish ventricles. Using newly generated gpnmb mutants, we analyze its role during heart regeneration and find that animals lacking gpnmb exhibit neutrophil retention and decreased macrophage recruitment as well as reduced myofibroblasts numbers. Moreover, loss of gpnmb impairs coronary endothelial cell regeneration and cardiomyocyte dedifferentiation. Transcriptomic analyses identified enhanced collagen production and extracellular matrix (ECM) related pathway activated in cryoinjured gpnmb mutant hearts. Furthermore, gpnmb mutant hearts exhibit larger fibrotic scars revealing defects in cardiac regeneration. Overall, these data suggest that gpnmb expressing macrophages modulate inflammation and ECM deposition after cardiac cryoinjury and highlight the importance of this subset of immune cells to support a regenerative response.
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Project description:Many risk loci for Parkinson’s disease (PD) have been identified by genome-wide association studies (GWAS), but target genes and mechanisms remain largely unknown. We linked the GWAS-derived chromosome 7 locus (sentinel SNP rs199347) to GPNMB, through colocalization analyses of expression quantitative trait locus (eQTL) and PD risk signals, confirmed by allele-specific expression studies in human brain. In cells, Glycoprotein Nonmetastatic Melanoma Protein B protein (GPNMB) co-immunoprecipitated and co-localized with alpha-synuclein (aSyn). In induced pluripotent stem cell-derived neurons, loss of GPNMB resulted in loss of ability to internalize aSyn fibrils and develop aSyn pathology. In 781 PD and 59 control biosamples, GPNMB was elevated in PD plasma, associating with disease severity. Thus, GPNMB represents a PD risk gene, with potential for biomarker development and therapeutic targeting.
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