anti trem 2  (R&D Systems)

Journal: International Journal of Molecular Sciences

Article Title: GM1 Oligosaccharide Modulates Microglial Activation and α-Synuclein Clearance in a Human In Vitro Model

doi: 10.3390/ijms26157634

Figure Lengend Snippet: Effects of GM1 oligosaccharide (OligoGM1) application on Human embryonic microglial (HCM3) cells. After 2 days of culture (80% of confluence), HCM3 cells were incubated or not incubated (control, CTRL) with OligoGM1 (100 µM). After 48 h, immunofluorescence staining [(against nuclei, ionized calcium-binding adaptor molecule 1 (Iba1) and triggered receptor expressed on myeloid cells 2 (TREM2)] and ELISA for quantification of released cytokines to medium were performed, as described in the : ( a ) Representative immunofluorescence images (20× magnification, scale bar 100 µm) and number of DAPI-positive cells; ( b ) Representative immunofluorescence images (20× magnification, scale bar 100 µm) of Iba1 expression, quantification of Iba1 accumulation (Iba1 area normalized by the number of cells), and area of Iba1-positive cells (area of microglia cells normalized by the µm 2 of Iba1); ( c ) Representative immunofluorescence images (20× magnification, scale bar 100 µm) of TREM2 expression, quantification of TREM2-positive cells (TREM2 area normalized by the number of cells), and area of TREM2-positive cells (area of microglia cells normalized by the µm 2 of TREM2); ( d ) Quantification of released cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). All values are represented as percentage versus CTRL and expressed as mean ± standard error of the mean (SEM, n = 6; ns = not significant, Mann–Whitney test).

Article Snippet: For immunofluorescence analyses, the following antibodies were used: primary mouse monoclonal anti-TREM2 antibody [Cat. 11084-MM08, research resource identifier (RRID):AB_2860315], purchased from Sino Biological Europe GmbH, Europe (Düsseldorfer, Germany); primary rabbit polyclonal anti-αSyn antibody (Cat. 2642S, RRID not available), purchased from Ozyme, Paris, France; and primary goat polyclonal anti-Iba1 antibody (Cat. Ab5076, RRID:AB_2224402), purchased from Abcam, Cambridge, UK.

Techniques: Incubation, Control, Immunofluorescence, Staining, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: GM1 Oligosaccharide Modulates Microglial Activation and α-Synuclein Clearance in a Human In Vitro Model

doi: 10.3390/ijms26157634

Figure Lengend Snippet: OligoGM1 modulation of αSyn-induced activation of HMC3 cells. After 2 days of culture (80% of confluence), HCM3 cells were incubated or not incubated (CTRL) with OligoGM1 (100 µM). After 4 h, αSyn (1 µM) was added to the culture medium for 48 h. At the end of treatment, immunofluorescence staining (against nuclei, Iba1, TREM2, and αSyn) and IL-6/TNF-α quantification from medium were performed as described in the . ( a ) Representative immunofluorescence images (20× magnification, scale bar 100 µm) and number of DAPI-positive cells; ( b ) Representative immunofluorescence images (20× magnification, scale bar 50 µm) of Iba1 expression, quantification of Iba1 accumulation (Iba1 area normalized by the number of cells), and area of Iba1-positive cells (area of microglia cells normalized by the µm 2 of Iba1); ( c ) Representative immunofluorescence images (20× magnification, scale bar 25 µm) of TREM2 and αSyn expressions, quantification of TREM2-positive cells (TREM2 area normalized by the number of cells), area of TREM2-positive cells (area of microglia cells normalized by the µm 2 of TREM2), and quantification of αSyn area accumulated within TREM(+) cells (area of αSyn normalized by the µm 2 of TREM2); ( d ) Quantification of released cytokines TNF-α and IL-6. All values are represented as percentage versus CTRL and expressed as mean ± SEM ( n = 6; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant by one-way ANOVA followed by Fisher’s LSD). * p < 0.05 was considered significant.

Article Snippet: For immunofluorescence analyses, the following antibodies were used: primary mouse monoclonal anti-TREM2 antibody [Cat. 11084-MM08, research resource identifier (RRID):AB_2860315], purchased from Sino Biological Europe GmbH, Europe (Düsseldorfer, Germany); primary rabbit polyclonal anti-αSyn antibody (Cat. 2642S, RRID not available), purchased from Ozyme, Paris, France; and primary goat polyclonal anti-Iba1 antibody (Cat. Ab5076, RRID:AB_2224402), purchased from Abcam, Cambridge, UK.

Techniques: Activation Assay, Incubation, Immunofluorescence, Staining, 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: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

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: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

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: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

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: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

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: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

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: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

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: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

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: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

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