Fig 1: TAMo specifically expressed EREG and AREG(A) Violin plots highlighting the differentially expressed genes in different clusters, as well as their potential functions. The TAMo-specific genes are highlighted in bold.(B) Developmental trajectory of monocytes and MDM clusters. The black curves represented the trajectory predicted by Slingshot.(C) Comparison of TAMo score in patients of different glioma grade and molecular subtype in the TCGA dataset. p values were calculated by Wilcoxon rank-sum test.(D) Representative images of GB samples simultaneously stained with CD45, CD14, EREG, and AREG antibodies. The arrowhead highlights cells with strong signals for all four channels. Scale bar, 20 μm.See also Figure S2.
Fig 2: TF activity analysis reveals FOSL2 as a regulator of TAMo(A) Heatmap of TF activity inferred by SCENIC. Each row represented the regulon of one TF, and each column represented one monocyte or MDM profiled by 10X. The cell subcluster and tissue of origin were marked on top of the heatmap.(B) The rank of feature importance scores of all TFs that potentially regulate EREG (top) and AREG expression (bottom). The dot size correlated with the importance of each TF, and the color represented whether the TF was over-expressed in GBM-infiltrating monocytes.(C) The presence of FOSL2-binding peaks and motifs located near EREG (left) and AREG (right) genes. The data was obtained from ENCODE data portal.(D and E) Expression levels of EREG and AREG in THP-1 monocyte cell line under FOSL2 over-expression (OE) (D) and knock-down (KD) (E) quantified by RT-PCR. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-sided unpaired t-test.(F) Boxplot of TAMo score across different anatomical regions in IvyGBM dataset. p values were calculated by Wilcoxon rank-sum test and between the highest group versus other samples.(G) Spatial distribution of Mono/Macro markers (CD14, ITGAM), FOSL2, TAMo score, and mesenchymal score across different histological regions in two IDH-wildtype GB patients. Abbreviations: PNZ, perinecrotic zone; PAN, pseudo-palisading cells around necrosis; MVP, microvascular proliferation; HBV, hyperplastic blood vessels; CT, cellular tumor; IT, infiltrating tumor; LE, leading edge.See also Figure S5.
Fig 3: TAMo promotes glioma invasion via the FOSL2-EREG/AREG-EGFR axis(A) Representative images of GB U251 cell in trans-well invasion assay under different treatment conditions. Scale bar, 100 μm. Abbreviations: CM, conditioned medium; EGFRi, EGFR inhibitor gefitinib.(B) Quantification of U251 invasiveness in the trans-well invasion assay. Data were presented as mean ± SD (n = 3 biological replicates). p values were calculated by two-sided unpaired t-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.(C) Quantification of mesenchymal markers including CD44 and TGFBI in U251 cells under different treatment conditions using RT-PCR assay (n = 4 biological replicates).(D) Representative images of GB U251 cell spheroid invasion 24 h after embedding in extracellular matrix with and without AREG and EREG treatment. From left to right: contour of the core (black lines) and invading protrusion of spheroids (green lines); raw images of spheroids, with the yellow squares representing the regions selected and zoomed in; zoomed view of spheroids, with white lines indicating the boundary between spheroid core and invading protrusion. Scale bar, 100 μm.(E) Quantification of GB cell invasion from spheroids. Ratio of invasion area represents the ratio of the protrusion area over the core area of each spheroid (n = 2 biological replicates for control and n = 3 biological replicates for other groups).(F) Proposed model of glioma- and/or hypoxia-mediated monocyte reprogramming into TAMo, which acted through the FOSL2-EREG/AREG-EGFR signaling axis and promoted mesenchymal transformation in glioma cells.See also Figure S6.
Supplier Page from MedChemExpress for Epiregulin Protein, Human