Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 1. Chronic TREM2 activation increases DAM around plaques. (A) Schematic of experimental design. 6-mo-old 5XFAD mice were injected with AD- tau in the HC (bregma: −2.5 mm; lateral: −2.0 mm; depth: −2.2 mm) and overlying cortex (bregma: −2.5 mm; lateral: −2.0 mm; depth: −1.0 mm) and sacrificed 3 mo later to evaluate peri-plaque pathologies. 1 wk before AD-tau injection and every week following until sacrifice, 5XFAD mice were given i.p. injections of 80 mg/ml of the AL002a mouse TREM2 antibody (n = 13 female, n = 14 male) or the IgG control antibody (n = 14 female, n = 12 male). (B) Quantification of TREM2 antibody levels in terminal plasma for 5XFAD mice either chronically treated with the IgG control antibody or the TREM2 antibody. (C) Quantification of TREM2 antibody levels in plasma for 5XFAD mice either acutely treated with the IgG control antibody or the TREM2 antibody. (D) Quantification of TREM2 antibody levels in cortical tissue for 5XFAD mice either acutely treated with the IgG control antibody or the TREM2 antibody. For acute treatment, there were n = 8 IgG control and n = 9 Trem2 antibody–treated age- and sex-matched mice, unless otherwise specified on graphs. (E) Representative images of ipsilateral hemisphere stained with CLEC7A+ microglia, P2RY12+ microglia, and Aβ from IgG control and TREM2 antibody–treated groups. Scale bars, 15 µm. (F) Quantification of mouse sTREM2 levels in plasma for 5XFAD mice either acutely treated with the IgG control antibody or the TREM2 antibody. (G) Quantification

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Injection, Control, Clinical Proteomics, Staining

Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 2. Chronic TREM2 activation with a TREM2 antibody results in no changes in Aβ plaque burden. (A) Representative images of Aβ plaques in 5XFAD mice either treated with the IgG control antibody or the TREM2 antibody. Scale bar, 100 µm. (B–E) Quantification of Aβ staining in the ipsi- and contralateral cortices (B and D) and hippocampi (C and E) in female mice. (F–I) Quantification of Aβ staining in the ipsi- and contralateral cortices (F and H) and hippocampi (G and I) in male mice. Triangle symbol represents female mice, and square symbol represents male mice either treated with the IgG control antibody (n = 14 female, n = 12 male) or the TREM2 antibody (n = 13 female, n = 14 male). Data are presented as mean ± SEM. Scale bar, 100 µm. Significance was determined using a Student’s t test. ns, P > 0.05.

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Control, Staining

Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 3. Chronic TREM2 activation with a TREM2 antibody increases NP-tau pathology. (A) Representative images of ipsi- and contralateral hemi- spheres stained with AT8+ NP-tau pathology in AD-tau injected 5XFAD mice either treated with the IgG control antibody or the TREM2 antibody. Repre- sentative images are from female mice. Scale bars, 100 µm. (B–E) Quantification of p-tau (AT8+) staining in the ipsi- and contralateral cortices (B and D) and hippocampi (C and E) in female mice. (F–I) Quantification of p-tau (AT8+) staining in the ipsi- and contralateral cortices (F and H) and hippocampi (G and I) in male mice. Triangle symbol represents female mice, and square symbol represents male mice either treated with the IgG control antibody (n = 14 female, n = 12 male) or the TREM2 antibody (n = 13 female, n = 14 male). Data are presented as mean ± SEM. Significance was determined using a Student’s t test. ns, P > 0.05; *, P < 0.05.

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Staining, Injection, Control

Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 4. Chronic TREM2 activation with a TREM2 antibody increases peri-plaque NP-tau pathology and plaque-associated neuritic dystrophy, and acute TREM2 activation results in no changes in AD-tau uptake and degradation. (A) Representative images of BACE1+ and X34+ staining in ipsilateral HC. Scale bars, 20 µm. (B) Representative images of AT8+ and X34+ staining in ipsilateral HC. (C–F) Quantification of the number of BACE1+ staining within 15 µm of plaques in the ipsi- and contra- cortices (C and E) and hippocampi (D and F). (G–J) Quantification of the number of AT8+ staining within 15 µm of plaques in the ipsi- and contra- cortices (G and I) and hippocampi (H and J). (K) AD-tau uptake assay in TREM2 WT BMDMs. Results represent two independent ex- periments. (L) AD-tau degradation assay in TREM2 WT BMDMs. Results represent two independent experiments. Triangle symbol represents female mice, and square symbol represents male mice either treated with the IgG control antibody (n = 14 female, n = 12 male) or the TREM2 antibody (n = 13 female, n = 14

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Staining, Degradation Assay, Control

Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 5. Chronic TREM2 activation with a TREM2 antibody increases peri-plaque loss of synaptic marker synapsin. (A) Representative images of Synapsin+ and X34+ staining in ipsilateral HC. Scale bar, 7 µm. (B) Quantification of the number of Synapsin+ puncta within 15 µm of plaques in the ipsi-HC. (C–E) Quantification of the number of Synapsin+ puncta within 15 µm of plaques in the ipsi-cortex, contra-cortex, and HC. Triangle symbol represents female mice, and square symbol represents male mice either treated with the IgG control antibody (n = 14 female, n = 12 male) or the TREM2 antibody (n = 13 female, n = 14 male). Data are presented as mean ± SEM. Significance was determined using a linear regression with sex as a covariate. ns, P > 0.05; *, P < 0.05.

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Marker, Staining, Control

Journal: Brain, behavior, and immunity

Article Title: Profiling TREM2 expression in amyotrophic lateral sclerosis.

doi: 10.1016/j.bbi.2023.01.013

Figure Lengend Snippet: Fig. 2. TREM2 mRNA expression profiling in human spinal cord in ALS. (A) The figure illustrates the maps of the 3 different TREM2 transcript variants. Black boxes represent exons, and arrows represent transcription start sites. (B) Overall TREM2 mRNA levels in the spinal cord in ALS samples (n = 21) were compared with controls (n = 19) as shown in the graph. (C) The dot-plot graph shows mRNA expression of the 3 TREM2 variants in human spinal cord specimens. (D–F) The panels show mRNA expression levels in ALS patients when compared with controls for each of the TREM2 transcript variant. Data represent the mean value ± SD. *p value < 0.05; **p value < 0.01; ***p value < 0.001; ****p value < 0.0001.

Article Snippet: CSF and serum levels of sTREM2 in ALS patients and controls were determined by a commercial enzyme-linked immunoabsorbent assay (ELISA), the Human TREM2 DuoSet ELISA (Catalog number: DY182805; R&D systems, Minneapolis, MN, USA) according to manufacturer’s instructions.

Techniques: Expressing, Variant Assay

Journal: Brain, behavior, and immunity

Article Title: Profiling TREM2 expression in amyotrophic lateral sclerosis.

doi: 10.1016/j.bbi.2023.01.013

Figure Lengend Snippet: Fig. 3. TREM2 protein expression is increased in spinal cord from ALS patients by western blot analysis. (A) Spinal cord samples from controls and ALS patients were loaded as labeled on top of lanes. β-actin expression is shown as reference control. (B) The bar chart represents the quantitative measurement of TREM2 relative to β-actin protein expression (n = 10). Data represent the mean value ± Standard Error of the Mean (SEM). **p value < 0.01.

Article Snippet: CSF and serum levels of sTREM2 in ALS patients and controls were determined by a commercial enzyme-linked immunoabsorbent assay (ELISA), the Human TREM2 DuoSet ELISA (Catalog number: DY182805; R&D systems, Minneapolis, MN, USA) according to manufacturer’s instructions.

Techniques: Expressing, Western Blot, Labeling, Control

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A , a diagram of the ex vivo mtROS and phagocytosis assay. B , Total amount of aortic F4/80 + /Trem2 + macrophages were quantified and shown in the bar graph; n=4-7 individual mice per group. C , MitoNeoD MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5 individual mice per group. D , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-6 individual mice per group. E , a diagram of the in vivo phagocytosis assay. F , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-7 individual mice per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Ex Vivo, Phagocytosis Assay, In Vivo

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A-D , Mouse scRNA-seq data were re-analyzed from a previous publication . Uniform manifold approximation and projection (UMAP) representation of 11 aortic CD45 + immune cell clusters were shown in A . Trem2 gene expression pattern ( B ) and Pkm gene expression pattern ( C ) were shown in the UMAP. D , Violin plots show the Pkm and Trem2 expression distribution among aortic macrophage subpopulations. ResMac: resident macrophages; InflaMac: inflammatory macrophages. E , HMDMs transfected with PKM siRNA were treated with 50 μg/ml oxLDL for 24 h before subjected to mtROS assay (left panel) or phagocytosis assay (right panel). MFI was quantified and shown in the bar graph; n=4-5 per group. F , Representative confocal images of macrophages immunostained for PKM2 (green) and Tom20 (red). Nuclei were stained by DAPI (blue). Scale bar: 5 μm. G , HMDMs treated with 20 μg/ml LDL (control) or oxLDL for 3 h were lysed, subjected to cell fractionation into mitochondrial and cytosol fractions. PKM2 and ATP5A (mitochondria fraction loading control) blot images from mitochondrial fractions were shown on the left. PKM2 and β-actin (cytosol fraction loading control) blot images from cytosol fractions were shown on the right. Images were quantified, normalized to each loading control, and expressed as fold change of control. n=4 per group. H , WT or Cd36 -null peritoneal macrophages treated with 20 μg/ml LDL (control) or oxLDL for 3 h and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and ATP5A and blot images were shown. Images were quantified, normalized, and expressed as fold change of control. n=4 per group. I , WT macrophages treated with 20 μg/ml oxLDL or pre-treated 1 or 5μM shikonin before addition of oxLDL, incubating for 3 h, and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and Tom20 and blot images were shown. Images were quantified and expressed as fold change of control. n=3 per group. J , WT macrophages pre-treated with 20 μg/ml oxLDL or in combination with 1 μM shikonin for 24 h before mtROS or phagocytosis assay. The MitoNeoD (left) or pHrodo (right) MFI was quantified and shown in the bar graph; n=3-4 per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Gene Expression, Expressing, Transfection, Phagocytosis Assay, Staining, Control, Cell Fractionation

Journal: Cell Death & Disease

Article Title: Adipocyte death triggers a pro-inflammatory response and induces metabolic activation of resident macrophages

doi: 10.1038/s41419-021-03872-9

Figure Lengend Snippet: A – C Expression of M1 markers CD11c, CD86, and CD9 and D , E M2 markers CD206 and CD301 by ATMs of induced CLS and interstitial macrophages. F , G Expression of CD64 (all macrophages) or TREM2. Targeted adipocytes are marked by an asterisk. M2 ATMs outside of CLS are highlighted by arrows. Scale bars = 50 µm.

Article Snippet: After fixation, the tissue was washed with PBS, blocked with staining buffer (3% bovine serum albumin (BSA) in PBS) for 1 h, and stained with pre-labeled antibodies in staining buffer (1:100 for antibodies from BioLegend, San Diego, USA: CD9 [Cat# 124810], CD36 [Cat# 102610], CD38 [Cat# 102716], CD11c [Cat# 117312], CD64 [Cat# 139332], CD86 [Cat# 105020], CD274 [Cat# 124312], F4/80 [Cat# 123122] and from R&DSystems, Minneapolis, USA: TREM2 [Cat# FAB17291A]; 1:200 for antibodies from AbD serotec, BioRad, Feldkirchen, Germany: CD301 [Cat# MCA2392A647] and CD206 [Cat# MCA2235A647T] at 4 °C overnight.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Adipocyte death triggers a pro-inflammatory response and induces metabolic activation of resident macrophages

doi: 10.1038/s41419-021-03872-9

Figure Lengend Snippet: Whole mount antibody staining of CLS formed in vivo in lean mice under homeostatic conditions. A – C ATMs in in vivo formed CLS express pro-inflammatory markers CD11c, CD86, and CD9, while interstitial ATMs are negative (highlighted by arrows). D , E Interstitial ATMs express the anti-inflammatory markers CD206 and CD301 (highlighted by arrows), while ATMs in CLS are negative. F , G Expression of CD64 (all macrophages) or TREM2 (highlighted by arrows). Asterisks mark adipocytes inside CLS. Scale bars = 100 µm.

Article Snippet: After fixation, the tissue was washed with PBS, blocked with staining buffer (3% bovine serum albumin (BSA) in PBS) for 1 h, and stained with pre-labeled antibodies in staining buffer (1:100 for antibodies from BioLegend, San Diego, USA: CD9 [Cat# 124810], CD36 [Cat# 102610], CD38 [Cat# 102716], CD11c [Cat# 117312], CD64 [Cat# 139332], CD86 [Cat# 105020], CD274 [Cat# 124312], F4/80 [Cat# 123122] and from R&DSystems, Minneapolis, USA: TREM2 [Cat# FAB17291A]; 1:200 for antibodies from AbD serotec, BioRad, Feldkirchen, Germany: CD301 [Cat# MCA2392A647] and CD206 [Cat# MCA2235A647T] at 4 °C overnight.

Techniques: Staining, In Vivo, Expressing

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Quantification of AR + cells in CD4 + T cells, CD8 + T cells, monocytes, and neutrophils from the peripheral blood of healthy individuals ( n = 31 biologically independent samples) and patients with prostate cancer ( n = 53 biologically independent samples) by flow cytometry. b Quantification of AR + cells in intraprostatic CD4 + T cells, CD8 + T cells, macrophages, and neutrophils was conducted in healthy prostates and prostate cancer tissues by flow cytometry ( n = 6 biologically independent samples). c , d The RNA-seq analysis of BMDMs cultured in RM1 CM and treated with ASC-J9 or DMSO. c A heatmap of DEGs in macrophages, where gene counts for the DMSO group have been normalised, and gene expression values are coloured based on upregulation (red) or downregulation (blue). DMSO treatment is represented in black, while ASC-J9 treatment is depicted in red. d A volcano plot displaying the gene expression of selected TREM family members ( Trem2 and Trem1 ), macrophage polarisation markers ( Cd163, Arg1, Cd86 , and Tnf ), and pro-migration factors ( Ccl2 and Ccl8 ), with gene expression values coloured according to upregulation (red) or downregulation (blue). e Quantification of TREM2 + cells in CD4 + T cells, CD8 + T cells, monocytes, and neutrophils from peripheral blood of healthy individuals ( n = 31 biologically independent samples) and patients with prostate cancer ( n = 53 biologically independent samples) by flow cytometry. f Quantification of TREM2 + cells in intraprostatic CD4 + T cells, CD8 + T cells, macrophages, and neutrophils was conducted in healthy prostates and prostate cancer tissues by flow cytometry ( n = 6 biologically independent samples). g Pearson correlation analysis of AR and TREM2 protein levels in peripheral blood monocytes of patients with prostate cancer ( n = 53 biologically independent samples). h Representative dot plots of TREM2 expression levels in peripheral blood mononuclear cells classified as TREM2 high (TREM2 high ), TREM2 low (TREM2 low ), and TREM2 negative (TREM2 neg ) (left). Representative dot plots of AR expression in peripheral blood TREM2 neg , TREM2 low , and TREM2 high mononuclear cells (middle). Quantification of AR expression in peripheral blood TREM2 neg , TREM2 low , and TREM2 high mononuclear cells of prostate cancer patients ( n = 53 biologically independent samples) (right). i Quantification of co-expression, singular expression, and non-expression of AR and TREM2 in peripheral monocytes of healthy individuals ( n = 31 biologically independent samples) and patients with prostate cancer ( n = 53 biologically independent samples). j Quantification of co-expression, singular expression, and non-expression of AR and TREM2 in intraprostatic macrophages of healthy prostate and prostate cancer tissues ( n = 6 biologically independent samples). k Representative immunoblot analysis of AR and TREM2 in CD68 + macrophages of tumour regions and adjacent normal prostate of prostate cancer patients. Experiment was repeated three times independently with similar results. l Representative multiplex immunofluorescence staining images of AR, TREM2, and CD206 in prostate tumour regions and adjacent normal prostate tissues. Nuclei were stained with DAPI. Scale bar: 10 μm. m Quantification of co-expression, singular expression, and non-expression of AR and TREM2 in CD206-expressing cells in tumour regions and distant normal prostate tissues ( n = 5 biologically independent samples) from multiplex immunofluorescence in (Fig. 1l). n Multiplex fluorescent immunohistochemistry (using TSA technology) analysis. Representative tumour regions of FFPE prostatectomy specimens were stained for CD68, CD206, CD86, AR, and TREM2. Each triangle or pentagon represents the CD68 + CD206 + cells or CD68 + CD86 + cells, respectively. Scale bar: 20 µm. o Percentage of TREM2 - AR - , TREM2 + AR - , TREM2 - AR + , and TREM2 + AR + cells in CD68 + CD206 + macrophages or CD68 + CD86 + macrophages in multiplex immunofluorescence image of the tumour regions of FFPE prostatectomy specimens, respectively ( n = 6 biologically independent samples). For ( l , n ) experiments were repeated three times independently with similar results. All the data are presented as mean ± SD. The P- values were determined by two-way ANOVA with Sidak’s multiple comparisons for ( a − o ); by the Wald test under a negative binomial generalized linear model, and adjusted for multiple testing via the Benjamini-Hochberg method for ( d ); by two-sided Pearson correlation analysis ( g ); and by one-way ANOVA with Tukey’s multiple comparisons for ( h ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: TREM2 (D8I4C) Rabbit mAb (Cell Signalling Technology, 91068, 1:1000), Androgen Receptor antibody (HUABIO, ER2001-34, 1:1000), Phospho-STAT3 (Tyr705) (D3A7) mAb (Cell Signalling Technology, 9145 T, 1:1000), Src (36D10) Rabbit mAb (Cell Signalling Technology, 2109, 1:1000), Phospho-Src (Tyr419) Antibody (Affinity Biosciences, AF3162, 1:500), Stat3 (79D7) Rabbit mAb (Cell Signalling Technology, 4904, 1:1000), Anti-Syk Antibody (Cell Signalling Technology, 2712, 1:1000), Phospho-Syk (Tyr525/526) (C87C1) mAb (Cell Signalling Technology, 2710, 1:1000), ROR gamma (t) mAb (AFKJS-9) (Thermo Fisher Scientific, 14-6988-80, 1:1000), Anti-beta-Actin mAb, Clone AC-74 (Sigma-Aldrich, A5316, 1:1000), Anti-Apolipoprotein E [ EPR19392 ] (Abcam, ab183597, 1:1000).

Techniques: Flow Cytometry, RNA Sequencing, Cell Culture, Gene Expression, Migration, Expressing, Western Blot, Multiplex Assay, Immunofluorescence, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Representative immunofluorescence staining images of AR and F4/80 in WT BMDMs and TREM2 KO BMDMs. Scale bar: 5 μm. b Representative flow plots and quantification of AR expression in WT BMDMs and TREM2 KO BMDMs ( n = 5 biologically independent samples). c Representative immunofluorescence staining images of AR in WT BMDMs treated with RM1 CM, RM1 CM + DMSO or RM1 CM + ENZA, respectively. Scale bar: 10 μm. d , e Protein expression levels of AR ( d ) and TREM2 ( e ) in WT BMDMs treated with DMSO or 10 μM ENZA for 48 h were quantified by flow cytometry ( n = 5 biologically independent samples). f , g Quantification of AR ( f ) and TREM2 ( g ) expression in WT BMDMs treated with RM1 CM for 48 h ( n = 5 biologically independent samples). h , i Relative protein expression of AR ( h ) and TREM2 ( i ) in THP1 cells treated with 22Rv1 CM for 48 h ( n = 5 biologically independent samples). j , k Protein expression levels of AR ( j ) and TREM2 ( k ) in THP1 cells treated with Lncap CM for 48 h ( n = 5 biologically independent samples). l , m Representative flow plots and quantification of TREM2 ( l ) and AR ( m ) in TAMs of Pten PC-/- TREM2 f/f mice and Pten PC-/- TREM2 f/f -Lyz2-cre mice ( n = 5 biologically independent samples). n The bubble chart of KEGG pathway enrichment analysis illustrated the significantly downregulated signalling pathways following ASC-J9 treatment in WT BMDMs. o The bubble chart of KEGG pathway enrichment analysis illustrated the significantly downregulated signalling pathways in RM1 CM treated TREM2 KO BMDMs compared to WT BMDMs. p Representative immunoblot analysis of AR, TREM2, p-STAT3, ROR-γ, p-Src, p-Syk, and p-JAK1 in WT BMDMs, TREM2 KO BMDMs and DAP12 KO BMDMs treated with RM1 CM for 48 h. Experiment was repeated three times independently with similar results. q Representative immunoblot analysis of AR and ROR-γ in WT BMDMs and TREM2 KO BMDMs treated with DMSO plus RM1 CM or 5 μM SR2211 (ROR-γ inhibitor) plus RM1 CM for 48 h. Experiment was repeated three times independently with similar results. r Representative immunoblot analysis of AR and p-STAT3 in WT BMDMs and TREM2 KO BMDMs treated with DMSO plus RM1 CM or 10 μM C188-9 (p-STAT3 inhibitor) plus RM1 CM for 48 h. Experiment was repeated three times independently with similar results. For a and c, experiments were repeated three times independently with similar results. All the data are presented as mean ± SD. The P- values were determined by two-sided Mann-Whitney U test for ( b , d − m ); and by Hypergeometric Test for ( n , o ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: TREM2 (D8I4C) Rabbit mAb (Cell Signalling Technology, 91068, 1:1000), Androgen Receptor antibody (HUABIO, ER2001-34, 1:1000), Phospho-STAT3 (Tyr705) (D3A7) mAb (Cell Signalling Technology, 9145 T, 1:1000), Src (36D10) Rabbit mAb (Cell Signalling Technology, 2109, 1:1000), Phospho-Src (Tyr419) Antibody (Affinity Biosciences, AF3162, 1:500), Stat3 (79D7) Rabbit mAb (Cell Signalling Technology, 4904, 1:1000), Anti-Syk Antibody (Cell Signalling Technology, 2712, 1:1000), Phospho-Syk (Tyr525/526) (C87C1) mAb (Cell Signalling Technology, 2710, 1:1000), ROR gamma (t) mAb (AFKJS-9) (Thermo Fisher Scientific, 14-6988-80, 1:1000), Anti-beta-Actin mAb, Clone AC-74 (Sigma-Aldrich, A5316, 1:1000), Anti-Apolipoprotein E [ EPR19392 ] (Abcam, ab183597, 1:1000).

Techniques: Immunofluorescence, Staining, Expressing, Flow Cytometry, Western Blot, MANN-WHITNEY

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Experimental scheme of mass spectrometry. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . Briefly, proteins from human prostate tumour tissues were extracted, followed by immunoprecipitation using anti-TREM2 antibody or IgG and agarose beads, and then the enriched proteins were lysed for peptide identification. b Venn diagram and the table showing the secretory proteins in the anti-TREM2-enriched complex. c Pearson correlation analysis of APOE and TREM2 mRNA levels in TCGA database of prostate cancer ( n = 498 biologically independent samples). d Representative immunoblot analysis of TREM2 and APOE in macrophages isolated from human prostate cancer tissues which was immunoprecipitated with anti-TREM2 antibody. Sample processing controls (lysate input, run on the same separate gel) are shown in panel (1 & 2). Experiment was repeated three times independently with similar results. e Representative multiplex immunofluorescence staining images of TREM2, APOE, and CD206 in prostate tumour tissues and adjacent normal prostate tissues. Nuclei were stained with DAPI. Scale bar: 10 μm. f Quantification of co-expression, singular expression, and non-expression of TREM2 and APOE in CD68-expressing cells in tumour regions and distant normal prostate tissues ( n = 5 biologically independent samples) from multiplex immunofluorescence of (Fig. 3e). g The concentration of APOE in the serum of both healthy individuals ( n = 12 biologically independent samples) and patients ( n = 28 biologically independent samples) with prostate cancer was detected using ELISA. h The concentration of APOE in the homogenates of normal prostate and prostate cancer tissues was detected using ELISA ( n = 6 biologically independent samples). i The concentration of APOE in normal cell culture media and RM1 CM was measured using ELISA ( n = 6 biologically independent samples). j Representative immunoblot analysis of AR, TREM2, p-STAT3, ROR-γ, p-Src, and p-Syk in WT BMDMs, TREM2 KO BMDMs and DAP12 KO BMDMs treated with RM1 CM or 100 nM recombinant APOE protein for 48 h. Experiment was repeated three times independently with similar results. k Representative immunoblot analysis of AR, TREM2, p-STAT3, ROR-γ, p-Src, and p-Syk in WT BMDMs treated with RM1 CM or RM1 CM plus 1 ng/ml anti-APOE for 48 h. Experiment was repeated three times independently with similar results. l , m RT-qPCR analysis of in WT BMDMs and TREM2 KO BMDMs treated with 100 nM recombinant APOE protein ( l ) or RM1 CM plus 1 ng/ml anti-APOE ( m ) for 48 h ( n = 6 biologically independent samples). Gene expression was normalized to Actb expression. For e, experiments were repeated three times independently with similar results. All the data are presented as mean ± SD. The P -values were determined by two-sided Pearson correlation analysis for ( c ); by two-way ANOVA with Sidak’s multiple comparisons for ( f ); by two-sided Mann-Whitney U test for ( g − i ); and by two-way ANOVA with Tukey’s multiple comparisons for ( l , m ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: TREM2 (D8I4C) Rabbit mAb (Cell Signalling Technology, 91068, 1:1000), Androgen Receptor antibody (HUABIO, ER2001-34, 1:1000), Phospho-STAT3 (Tyr705) (D3A7) mAb (Cell Signalling Technology, 9145 T, 1:1000), Src (36D10) Rabbit mAb (Cell Signalling Technology, 2109, 1:1000), Phospho-Src (Tyr419) Antibody (Affinity Biosciences, AF3162, 1:500), Stat3 (79D7) Rabbit mAb (Cell Signalling Technology, 4904, 1:1000), Anti-Syk Antibody (Cell Signalling Technology, 2712, 1:1000), Phospho-Syk (Tyr525/526) (C87C1) mAb (Cell Signalling Technology, 2710, 1:1000), ROR gamma (t) mAb (AFKJS-9) (Thermo Fisher Scientific, 14-6988-80, 1:1000), Anti-beta-Actin mAb, Clone AC-74 (Sigma-Aldrich, A5316, 1:1000), Anti-Apolipoprotein E [ EPR19392 ] (Abcam, ab183597, 1:1000).

Techniques: Mass Spectrometry, Immunoprecipitation, Western Blot, Isolation, Multiplex Assay, Immunofluorescence, Staining, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Recombinant, Quantitative RT-PCR, Gene Expression, MANN-WHITNEY

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Schematic representation of TREM2 +/+ and TREM2 -/- BMDMs induction. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . BMDMs were treated with RM1 CM plus 10 μM ENZA, and gene expression was analysed by RT-qPCR and protein expression by flow cytometry. b RT-qPCR analysis of M2-like macrophage markers Il10 and Tgfb1 in WT BMDMs and TREM2 KO BMDMs treated with RM1 CM plus 10 μM ENZA for 48 h ( n = 6 biologically independent samples). Gene expression was normalized to Actb expression. c ELISA analysis of IL-10 and TGF-β in the supernatant of WT BMDMs and TREM2 KO BMDMs treated with RM1 CM plus 10 μM ENZA for 48 h ( n = 5 biologically independent samples). d , e Representative flow plots and quantification of CD206 ( d ) and CD86 ( e ) in WT BMDMs and TREM2 KO BMDMs treated with RM1 CM plus 10 μM ENZA for 48 h ( n = 5 biologically independent samples). f A genomic view of AR enrichment on IL10 and TGFB1 promoters in THP-1 cells was analysed from published ChIP-seq data . AR peaks under DMSO and 10 nM R1881 conditions are depicted in blue and red, respectively. g Prediction of Ar binding sites on the Il10 (left) and Tgfb1 (right) promoters through the JASPAR database. h The specific binding of Ar to the predicted binding site in Il10 and Tgfb1 promoters in WT BMDMs treated with RM1 CM was detected by ChIP-qPCR ( n = 6 biologically independent samples). i The binding of Ar to the Il10 and Tgfb1 promoters in WT BMDMs and TREM2 KO BMDMs treated with RM1 CM was detected by ChIP-qPCR ( n = 6 biologically independent samples). Relative fold enrichment was normalised relative to IgG and WT BMDM group, respectively. j The binding of Ar to the Il10 and Tgfb1 promoters in WT BMDMs treated with RM1 CM plus 10 μM ENZA for 48 h was detected by ChIP-qPCR ( n = 6 biologically independent samples). Relative fold enrichment was normalised relative to IgG and CTL group, respectively. All the data are presented as mean ± SD. The P -values were determined by two-way ANOVA with Tukey’s multiple comparisons for ( b − d ); by two-way ANOVA with Sidak’s multiple comparisons for ( h ); and by two-sided Mann-Whitney U test for ( i , j ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: TREM2 (D8I4C) Rabbit mAb (Cell Signalling Technology, 91068, 1:1000), Androgen Receptor antibody (HUABIO, ER2001-34, 1:1000), Phospho-STAT3 (Tyr705) (D3A7) mAb (Cell Signalling Technology, 9145 T, 1:1000), Src (36D10) Rabbit mAb (Cell Signalling Technology, 2109, 1:1000), Phospho-Src (Tyr419) Antibody (Affinity Biosciences, AF3162, 1:500), Stat3 (79D7) Rabbit mAb (Cell Signalling Technology, 4904, 1:1000), Anti-Syk Antibody (Cell Signalling Technology, 2712, 1:1000), Phospho-Syk (Tyr525/526) (C87C1) mAb (Cell Signalling Technology, 2710, 1:1000), ROR gamma (t) mAb (AFKJS-9) (Thermo Fisher Scientific, 14-6988-80, 1:1000), Anti-beta-Actin mAb, Clone AC-74 (Sigma-Aldrich, A5316, 1:1000), Anti-Apolipoprotein E [ EPR19392 ] (Abcam, ab183597, 1:1000).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, ChIP-sequencing, Binding Assay, ChIP-qPCR, MANN-WHITNEY

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a , b Assessment of the indirect antitumour effects of BMDMs. WT BMDMs and TREM2 KO BMDMs were co-cultured with RM1 cells at a ratio of 1:1 in Transwell systems with 10 μM ENZA. a RT-qPCR was performed to detect the mRNA levels of Ki67 in RM1 cells after 48 h of co-culture ( n = 6 biologically independent samples). b Representative images and quantification of migrated RM1 cells were obtained after 24 h of co-culture ( n = 5 biologically independent samples). c , d WT BMDMs and TREM2 KO BMDMs were co-cultured with RM1 cells at a ratio of 1:1 in Transwell systems with 100 nM recombinant APOE protein. c RT-qPCR was performed to detect the mRNA levels of Ki67 in RM1 cells after 48 h of co-culture ( n = 6 biologically independent samples). d Representative images and quantification of migrated RM1 cells were obtained after 24 h of co-culture ( n = 5 biologically independent samples). e RT-qPCR analysis of pro-migratory cytokines Ccl2 and pro-proliferative cytokine Il23a in WT BMDMs and TREM2 KO BMDMs co-cultured with RM1 cells plus 10 μM ENZA for 48 h ( n = 6 biologically independent samples). Gene expression was normalized to Actb expression. f Representative images of Transwell migration assays (with three technical replicates) of RM1 cells cultured in normal medium alone or co-cultured with WT BMDMs at a ratio of 1:1, or co-cultured with WT BMDMs and treated with 1 ug/ml blocking antibodies against CCL2, CCL7, and CCL13. Cells that penetrated the membrane after 24 h of culture were stained using crystal violet. The migration ability of the cells was quantified using the optical density (OD) of the crystal violet-stained cells ( n = 3 independent experiments). The schematics in ( a − d , f ) are created in Biorender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . g WT BMDMs were co-cultured with RM1 cells at a ratio of 1:1 plus blocking antibody against Ki67 in Transwell systems for 48 h, RT-qPCR was performed to detect the mRNA levels of Ki67 in RM1 cells ( n = 6 biologically independent samples). h ELISA analysis of CCL2 and IL-23 in the supernatant of WT BMDMs and TREM2 KO BMDMs treated with RM1 CM plus 10 μM ENZA for 48 h ( n = 5 biologically independent samples). i A genomic view of AR enrichment on IL23A and CCL2 promoters in THP-1 cells was analysed from published ChIP-seq data . AR peaks under DMSO and 10 nM R1881 conditions are depicted in blue and red, respectively. j Prediction of AR binding sites on the Ccl2 (left) and Il23a (right) promoters through the JASPAR database. k The specific binding of Ar to the predicted binding site in Ccl2 and Il23a promoters in WT BMDMs treated with RM1 CM was detected by ChIP-qPCR ( n = 6 biologically independent samples). l The binding of Ar to the Ccl2 and Il23a promoters in WT BMDMs and TREM2 KO BMDMs treated with RM1 CM was detected by ChIP-qPCR ( n = 6 biologically independent samples). Relative fold enrichment was normalised relative to IgG and WT BMDM group, respectively. m The binding of Ar to the Ccl2 and Il23a promoters in WT BMDMs treated with RM1 CM plus 10 μM ENZA for 48 h was detected by ChIP-qPCR ( n = 6 biologically independent samples). Relative fold enrichment was normalised relative to IgG and CTL group, respectively. All the data are presented as mean ± SD. The P -values were determined by two-way ANOVA with Tukey’s multiple comparisons for ( a − e , h ); by two-way ANOVA with Sidak’s multiple comparisons for ( k ); by one-way ANOVA with Tukey’s multiple comparisons for ( f ); and by two-sided Mann-Whitney U test for ( g − m ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: TREM2 (D8I4C) Rabbit mAb (Cell Signalling Technology, 91068, 1:1000), Androgen Receptor antibody (HUABIO, ER2001-34, 1:1000), Phospho-STAT3 (Tyr705) (D3A7) mAb (Cell Signalling Technology, 9145 T, 1:1000), Src (36D10) Rabbit mAb (Cell Signalling Technology, 2109, 1:1000), Phospho-Src (Tyr419) Antibody (Affinity Biosciences, AF3162, 1:500), Stat3 (79D7) Rabbit mAb (Cell Signalling Technology, 4904, 1:1000), Anti-Syk Antibody (Cell Signalling Technology, 2712, 1:1000), Phospho-Syk (Tyr525/526) (C87C1) mAb (Cell Signalling Technology, 2710, 1:1000), ROR gamma (t) mAb (AFKJS-9) (Thermo Fisher Scientific, 14-6988-80, 1:1000), Anti-beta-Actin mAb, Clone AC-74 (Sigma-Aldrich, A5316, 1:1000), Anti-Apolipoprotein E [ EPR19392 ] (Abcam, ab183597, 1:1000).

Techniques: Cell Culture, Quantitative RT-PCR, Co-Culture Assay, Recombinant, Gene Expression, Expressing, Migration, Blocking Assay, Membrane, Staining, Enzyme-linked Immunosorbent Assay, ChIP-sequencing, Binding Assay, ChIP-qPCR, MANN-WHITNEY

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Box plot depicting AR (left), TREM2 (medium) and APOE (right) expression in TCGA bulk RNA-seq samples of human normal prostate ( n = 152 biologically independent samples) and prostate cancer samples (PRAD, n = 492 biologically independent samples) from GEPIA database. Box plots show the median (centre line), 25th and 75th percentiles (box bounds), and whiskers extend to the 5th and 95th percentiles which represent minima and maxima. b Disease-free survival based on AR, TREM2 and APOE expression in primary prostate tumours (TCGA) from GEPIA database. the High AR (left) /TREM2 (medium) /APOE (right) group (red) ( n = 246 biologically independent samples) corresponds to the first 50% of expression, and the Low AR (left) /TREM2 (medium) /APOE (right) group (blue) ( n = 246 biologically independent samples) corresponds to the last 50% of expression. c Experimental scheme. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . C57BL/6 TREM2 f/f and TREM2 f/f -Lyz2-cre mice were subjected to subcutaneous injections of RM1-Luc cells in the inguinal region. Surgical castration (CTX) was performed after 3 days. ENZA treatment commenced on day five. Mice were euthanized on day 15 for analysis and data collection. The sham group for each genotype serves as its own control. RM1-Luc: luciferase-tagged RM1, s.c.: subcutaneous, CTX: Castration, ENZA: Enzalutamide. d − f Representative prostate tumour size ( n = 5 mice) ( d ), tumour weight ( n = 6 mice) ( e ), and tumour growth curve ( n = 5 mice) ( f ) of each group. g The proportion of CD45 + CD11b + F4/80 + macrophages in the tumour were quantified by flow cytometry ( n = 5 mice). h The proportion of CD11b + F4/80 + CD206 + (left) macrophages and CD11b + F4/80 + CD86 + (right) macrophages in the tumour were quantified by flow cytometry ( n = 5 mice). i ELISA assay was employed to quantify the protein levels of IL-10 and TGF-β in the tumour griding supernatant of tumour-bearing mice ( n = 5 mice). j The proportion of CD45 + CD3 + CD8 + T cells in the tumour was quantified by flow cytometry ( n = 5 mice). k The number of PD1 + CD8 + T cells mg -1 in the tumour ( n = 5 mice). l The number of IFN-γ + CD8 + T cells mg -1 (left) and TNF + CD8 + T cells mg -1 (right) in the tumour ( n = 5 mice). m The proportion of granzyme B (left) and perforin (right) in the intraprostatic CD8 + T cells of tumour-bearing mice ( n = 5 mice). (n) Experimental scheme. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . C57BL/6 TREM2 f/f and TREM2 f/f -Lyz2-cre mice were subjected to subcutaneous injections of RM1 cells in the inguinal region. Surgical castration (CTX) was performed after 3 days. ENZA and anti-PD1 treatment commenced on day five. Mice were euthanized on day 15 for analysis and data collection. o , p Representative prostate tumour size ( n = 6 mice) ( o ), and tumour weight ( n = 6 mice) ( p ) of each group. All the data are presented as mean ± SD. The P -values were determined by two-sided Mann-Whitney U test for ( a ); by Log-rank (Mantel-Cox) test for ( b ); by two-way ANOVA with Tukey’s multiple comparisons for ( f ); and by one-way ANOVA with Tukey’s multiple comparisons for ( e , g − m , p ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: TREM2 (D8I4C) Rabbit mAb (Cell Signalling Technology, 91068, 1:1000), Androgen Receptor antibody (HUABIO, ER2001-34, 1:1000), Phospho-STAT3 (Tyr705) (D3A7) mAb (Cell Signalling Technology, 9145 T, 1:1000), Src (36D10) Rabbit mAb (Cell Signalling Technology, 2109, 1:1000), Phospho-Src (Tyr419) Antibody (Affinity Biosciences, AF3162, 1:500), Stat3 (79D7) Rabbit mAb (Cell Signalling Technology, 4904, 1:1000), Anti-Syk Antibody (Cell Signalling Technology, 2712, 1:1000), Phospho-Syk (Tyr525/526) (C87C1) mAb (Cell Signalling Technology, 2710, 1:1000), ROR gamma (t) mAb (AFKJS-9) (Thermo Fisher Scientific, 14-6988-80, 1:1000), Anti-beta-Actin mAb, Clone AC-74 (Sigma-Aldrich, A5316, 1:1000), Anti-Apolipoprotein E [ EPR19392 ] (Abcam, ab183597, 1:1000).

Techniques: Expressing, RNA Sequencing, Control, Luciferase, Flow Cytometry, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Experimental scheme. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . RM1-Luc cells were intra-prostatically injected into C57BL/6 TREM2 f/f and TREM2 f/f -Lyz2-cre mice. Surgical castration was performed after 3 days. ENZA treatment commenced on day five. IVIS analysis of mice was performed every 3 days starting from day eight. Mice were euthanized on day 17 for analysis and data collection. b Survival curve of mice intra-prostatically injected with RM1-Luc cells ( n = 10 mice). c Representative IVIS fluorescence images and ROI quantification of prostate tumours in mice of each group ( n = 3 mice). d Representative IVIS fluorescence images and ROI quantification of the lymph node, lung, and liver in tumour-bearing mice ( n = 3 mice). e Experimental scheme. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . RM1-Luc cells were subcutaneously injected into C57BL/6 TREM2 f/f and TREM2 f/f -Lyz2-cre mice. Radical resection was performed to remove the tumour on day three. Surgical castration was performed on day five. IVIS analysis of mice was conducted every 5 days starting from day eight. Mice were euthanized on day 18 for analysis and data collection. f , g Representative recurrent prostate tumour images ( f ) and tumour weight ( g ) of each group ( n = 5 mice). h Representative IVIS fluorescence images and ROI quantification of recurrent prostate tumours in mice ( n = 3 mice). i Experimental scheme. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . BMDMs from C57BL/6 TREM2 +/+ CD45.2 mice and C57BL/6 TREM2 -/- CD45.2 mice were injected into CD45.1 mice via the tail vein, and RM1-Luc cells were subcutaneously injected into the inguinal region of the mice 24 h later. Surgical castration was performed after 3 days. ENZA treatment commenced on day five. IVIS analysis of mice was performed every 4 days starting from day seven. Mice were euthanized on day 15 for analysis and data collection. j , k Representative prostate tumour images ( j ) and tumour weight ( k ) of each group ( n = 5 mice). RM1-Luc: luciferase-tagged RM1, s.c.: subcutaneous, CTX: Castration, ENZA: Enzalutamide, IVIS: In vivo imaging system, BMDM: Bone marrow-derived macrophages. All the data are presented as mean ± SD. The P -values were determined by Log-rank (Mantel-Cox) test for ( b ); by two-way ANOVA with Tukey’s multiple comparisons for ( c , h ); and by one-way ANOVA with Tukey’s multiple comparisons for ( d − k ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: TREM2 (D8I4C) Rabbit mAb (Cell Signalling Technology, 91068, 1:1000), Androgen Receptor antibody (HUABIO, ER2001-34, 1:1000), Phospho-STAT3 (Tyr705) (D3A7) mAb (Cell Signalling Technology, 9145 T, 1:1000), Src (36D10) Rabbit mAb (Cell Signalling Technology, 2109, 1:1000), Phospho-Src (Tyr419) Antibody (Affinity Biosciences, AF3162, 1:500), Stat3 (79D7) Rabbit mAb (Cell Signalling Technology, 4904, 1:1000), Anti-Syk Antibody (Cell Signalling Technology, 2712, 1:1000), Phospho-Syk (Tyr525/526) (C87C1) mAb (Cell Signalling Technology, 2710, 1:1000), ROR gamma (t) mAb (AFKJS-9) (Thermo Fisher Scientific, 14-6988-80, 1:1000), Anti-beta-Actin mAb, Clone AC-74 (Sigma-Aldrich, A5316, 1:1000), Anti-Apolipoprotein E [ EPR19392 ] (Abcam, ab183597, 1:1000).

Techniques: Injection, Fluorescence, Luciferase, In Vivo Imaging, Derivative Assay

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Experimental scheme. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . Pten PC-/- TREM2 f/f and Pten PC-/- TREM2 f/f -Lyz2-cre mice were surgically castrated at week eight. ENZA was administrated 12 weeks after surgical castration when castration resistance was observed. Prostate tumour volume was monitored by magnetic resonance imaging (MRI) at weeks 12 and 24. Mice were euthanized at week 24 for analysis and data collection. CTX: Castration, ENZA: Enzalutamide, MRI: Magnetic resonance imaging. b Representative MRI scans of mice in each group at weeks 12 and 24. The red circle highlights the area of prostate carcinoma. c Waterfall plots depicting the proportional change in tumour response at weeks 12 and 24 ( n = 3 mice). d Representative haematoxylin and eosin (H&E) and Ki67 staining at the observation endpoint. Scale bar: 40 μm. e Quantitative analysis of adenocarcinoma, prostatic intraepithelial neoplasia (PIN), or normal-like glands at the observation endpoint in each group ( n = 3 mice). f Percentage of Ki-67 + cell area relative to total area ( n = 3 mice). g ELISA assay was employed to quantify the protein levels of IL-10 (left) and TGF-β (right) in tumour griding supernatant of mice ( n = 5 mice). h The proportion of CD45 + CD11b + F4/80 + macrophages in the tumour were quantified by flow cytometry ( n = 5 mice). i The proportion of CD11b + F4/80 + CD206 + (up) macrophages and CD11b + F4/80 + CD86 + (down) macrophages in the tumour were quantified by flow cytometry ( n = 5 mice). j The proportion of CD45 + CD3 - NK1.1 + NK cells in the tumour was quantified by flow cytometry ( n = 5 mice). k The proportion of CD45 + CD3 + CD8 + T cells in the tumour was quantified by flow cytometry ( n = 5 mice). l The number of PD1 + CD8 + T cells mg -1 in the tumour ( n = 5 mice). m The number of IFN-γ + CD8 + T cells mg -1 (left) and TNF + CD8 + T cells mg -1 (right) in the tumour ( n = 5 mice). n The number of Granzyme B + CD8 + T cells mg -1 (left) and Perforin + CD8 + T cells mg -1 (right) in the tumour ( n = 5 mice). For b and d, experiments were repeated three times independently with similar results. All the data are presented as mean ± SD. The P- values were determined by two-way ANOVA with Tukey’s multiple comparisons for (c, e); and by one-way ANOVA with Tukey’s multiple comparisons for ( f – n ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: TREM2 (D8I4C) Rabbit mAb (Cell Signalling Technology, 91068, 1:1000), Androgen Receptor antibody (HUABIO, ER2001-34, 1:1000), Phospho-STAT3 (Tyr705) (D3A7) mAb (Cell Signalling Technology, 9145 T, 1:1000), Src (36D10) Rabbit mAb (Cell Signalling Technology, 2109, 1:1000), Phospho-Src (Tyr419) Antibody (Affinity Biosciences, AF3162, 1:500), Stat3 (79D7) Rabbit mAb (Cell Signalling Technology, 4904, 1:1000), Anti-Syk Antibody (Cell Signalling Technology, 2712, 1:1000), Phospho-Syk (Tyr525/526) (C87C1) mAb (Cell Signalling Technology, 2710, 1:1000), ROR gamma (t) mAb (AFKJS-9) (Thermo Fisher Scientific, 14-6988-80, 1:1000), Anti-beta-Actin mAb, Clone AC-74 (Sigma-Aldrich, A5316, 1:1000), Anti-Apolipoprotein E [ EPR19392 ] (Abcam, ab183597, 1:1000).

Techniques: Magnetic Resonance Imaging, Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry