Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mutant p53 epigenetically rewires CXCL10 to promote CD8⁺ T-cell infiltration and enhance the anti-PD-1 response in advanced prostate cancer

doi: 10.1186/s13046-026-03672-z

Figure Lengend Snippet: Trp53 p.R245Q promotes prostate tumor growth and remodels the tumor microenvironment. a Schematic of CRISPR/Cas9-mediated knock-in of Trp53 p.R245Q (arginine→glutamine) in prostate cancer cells and the workflow for in vivo validation and single-cell RNA sequencing. b-d Subcutaneous tumor growth of Myc-CaP cells in FVB/NJ mice (1 × 10 6 cells, WTp53 n = 5; Mutp53 n = 5): representative images of tumors at the experimental endpoint ( b ), tumor growth curves ( c ), and endpoint tumor weights ( d ). Tumors derived from Mutp53-expressing Myc-CaP cells exhibited significantly accelerated growth compared with those derived from control cells ( p < 0.05). e-g Subcutaneous tumor growth of RM-1 cells (1 × 10 6 cells in a Matrigel/PBS mixture) in C57BL/6 mice: representative endpoint tumor images ( e ), longitudinal tumor volume curves ( f ), and final tumor weights at sacrifice ( g ). Compared with those derived from WTp53 control cells, the tumors derived from Mutp53-expressing RM-1 cells markedly accelerated progression (WTp53 n = 5; Mutp53 n = 5; p < 0.05). h Kaplan-Meier survival analysis of subcutaneous RM-1 tumor-bearing mice injected with 5 × 10 5 cells: Compared with WTp53 controls, Mutp53-bearing mice presented significantly shorter overall survival (WTp53 n = 8; Mutp53 n = 10; log-rank test, p = 0.0156). i Dot plot visualization of canonical marker gene expression across major cell types in the single-cell transcriptome dataset derived from orthotopic RM-1 prostate tumors (5 × 10 5 cells implanted), including epithelial cells, endothelial cells, monocytes/macrophages, T/NK cells, fibroblasts, and pericytes, used for cell type annotation. j UMAP visualization of single-cell transcriptomes showing the distribution of major annotated cell types, along with representative top differentially expressed gene modules. k Fractional abundance of major cell populations in the tumor microenvironment. Box plots showing the relative fractions of epithelial cells, endothelial cells, pericytes, monocytes/macrophages, fibroblasts, and T/NK cells in tumors from the WTp53 and Mutp53 groups derived from single-cell transcriptome analysis. Among these populations, fibroblasts were obviously reduced in Mutp53 tumors, whereas changes in other cell types did not reach statistical significance. l Heatmap of observed-to-expected ratios for major cell populations. The Ro/e ratio was calculated for fibroblasts, pericytes, epithelial cells, monocytes/macrophages, endothelial cells, and T/NK cells in WTp53 and Mutp53 tumors. Compared with WTp53 tumors, Mutp53 tumors presented reduced fibroblast and pericyte enrichment, whereas monocytes/macrophages were relatively enriched. m Multiplex immunofluorescence staining of tumor sections for Pan-CK, CD4, FoxP3, CD8, CD68, CD163, and CTLA-4; Mutp53 tumors exhibit increased infiltration of CD4 + FoxP3+ regulatory T cells, CD8 + cytotoxic T cells, and CD68+/CD163 + macrophages, alongside elevated CTLA-4 expression. Scale bars: 100 μm. Note: Unless otherwise specified, p < 0.05 was considered statistically significant. Significance levels are indicated as follows: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Abbreviations: WTp53: Wild-type p53; Mutp53: Mutant p53 ( Trp53 p.R245Q, arginine to glutamine substitution); CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; Cas9: CRISPR-associated protein 9; scRNA-seq: Single-cell RNA sequencing; UMAP: Uniform Manifold Approximation and Projection; Ro/e: Ratio of observed-to-expected frequency; Mon/Macro: Monocytes/Macrophages; T/NK cells: T lymphocytes/Natural Killer cells; Fibro: Fibroblasts; Peri: Pericytes; Endo: Endothelial cells; Epi: Epithelial cells

Article Snippet: The mouse prostate cancer cell lines RM-1 (CRL-3310TM) and MyC-CaP (CRL-3255TM) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which provides authenticated lines originally sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: CRISPR, Knock-In, In Vivo, Biomarker Discovery, Single Cell, RNA Sequencing, Derivative Assay, Expressing, Control, Injection, Marker, Gene Expression, Multiplex Assay, Immunofluorescence, Staining, Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mutant p53 epigenetically rewires CXCL10 to promote CD8⁺ T-cell infiltration and enhance the anti-PD-1 response in advanced prostate cancer

doi: 10.1186/s13046-026-03672-z

Figure Lengend Snippet: Mutant p53 tumors exhibit enhanced sensitivity to PD-1 blockade through augmented CD8 + cytotoxic programs. a Schematic of the experimental design for the subcutaneous and orthotopic RM-1 tumor models treated with an anti-PD-1 antibody. b-d Subcutaneous tumor growth of WTp53 and Mutp53 RM-1 tumors established by injection of 1 × 10 6 cells, with or without anti-PD-1 therapy: representative images ( b ), tumor volume curves ( c ), and endpoint tumor weights ( d ) ( n = 5 per group, one-way ANOVA with Tukey’s test). e-g Orthotopic tumor growth of WTp53 and Mutp53 RM-1 tumors established by prostate implantation of 5 × 10 5 cells following PD-1 blockade: representative images ( e ), tumor volumes ( f ), and endpoint tumor weights ( g ) ( n = 4–5 per group, one-way ANOVA with Tukey’s test). h-i Flow cytometric analysis of intratumoral CD8 + GZMB + T cells at baseline and after PD-1 blockade; quantification is shown in (I) (one-way ANOVA with Tukey’s test). j-k Single-cell RNA-seq clustering of CD8 + T-cell subsets, highlighting enrichment of CD8+, CD8 + CD69+, CD8 + GZMA+GZMB+, CD8 + NKG7+, and CD4 + CD8+ T cells in Mutp53 tumors relative to WTp53 tumors. l-s Functional module scoring of CD8 + T cells, showing comparable costimulatory ( l-m ), exhausted ( n-o ), and resident ( r-s ) signatures between groups but markedly increased cytotoxic signatures in Mutp53 tumors ( p-q ). t Expression of representative effector genes in intratumoral CD8 + T cells, showing upregulation of Nkg7 , Gzmb , and Slamf7 in Mutp53 tumors, whereas Ifng expression remained unchanged (see also Fig. S4a). u Multiplex immunofluorescence staining for CD8 and GZMB, showing increased infiltration of CD8 + GZMB + T cells in Mutp53 tumors at baseline and after PD-1 blockade, alongside reduced IFN-γ expression, compared with WTp53 (see also Fig. S4b-c). Note: Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant. Abbreviations: Mutp53, mutant p53; WTp53, wild-type p53; PD-1, programmed cell death protein-1; scRNA-seq, single-cell RNA sequencing; GZMA, granzyme A; GZMB, granzyme B; IFN-γ, interferon-γ; UMAP, uniform manifold approximation and projection

Article Snippet: The mouse prostate cancer cell lines RM-1 (CRL-3310TM) and MyC-CaP (CRL-3255TM) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which provides authenticated lines originally sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Mutagenesis, Injection, Single Cell, RNA Sequencing, Functional Assay, Expressing, Multiplex Assay, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mutant p53 epigenetically rewires CXCL10 to promote CD8⁺ T-cell infiltration and enhance the anti-PD-1 response in advanced prostate cancer

doi: 10.1186/s13046-026-03672-z

Figure Lengend Snippet: Mutp53 promotes immunotherapy responsiveness through CXCL10-CXCR3-mediated recruitment and activation of CD8 + T cells. a-b Heatmap ( a ) and qRT-PCR analysis ( b ) of immune-related chemokine expression in subcutaneous WTp53 and Mutp53 tumors, showing significant upregulation of Cxcl10 together with Cxcl1 , Cxcl2 , Cxcl12 , Ccl2 , and Cxcl9 in Mutp53 tumors compared with WTp53 tumors. c Correlation analysis of CXCL10 expression with immune-related signaling pathways in the IMvigor210 urothelial carcinoma cohort treated with immune checkpoint blockade, revealing strong positive associations with IFN-γ-related signatures and the antigen presentation machinery (APM). d Representative immunohistochemical (IHC) staining of CXCL10 in WTp53 and Mutp53 tumors. e-f Immunoblot analysis ( e ) and quantitative densitometry ( f ) of CXCL10 protein expression in WTp53 and Mutp53 tumor cell lines and corresponding tumor tissues. g Transwell coculture assays demonstrating enhanced chemotaxis of CD8 + T cells toward Mutp53-derived tumor cells compared with WTp53-derived tumor cells ( n = 3). h Clinical association between intratumoral CXCL10 expression and immune checkpoint blockade (ICB) benefit in the IMvigor210 cohort, with higher CXCL10 levels predicting improved therapeutic response. i Schematic illustration of the in vivo experimental design for pharmacological disruption of the CXCL10-CXCR3 axis using the CXCR3 antagonist AMG-487 during anti-PD-1 treatment, with RM-1 tumors established by subcutaneous injection of 1 × 10⁶ cells. j-l Representative tumor images ( j ), tumor growth curves ( k ), and endpoint tumor weights ( l ) of WTp53- and Mutp53-bearing mice treated with anti-PD-1 therapy in the presence or absence of AMG-487 ( n = 5 per group). CXCR3 blockade markedly attenuated the therapeutic efficacy of PD-1 blockade, with a substantially stronger reversal observed in Mutp53 tumors. m Serum biochemical analyses evaluating liver and kidney function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (BUN), and creatinine (CREA), showing no significant differences among treatment groups, indicating minimal systemic toxicity of AMG-487. n Multiplex immunofluorescence staining of tumor sections for CD8 (green), granzyme B (GZMB; red), and nuclei (DAPI; blue), revealing that anti-PD-1 therapy markedly increased infiltration of GZMB+CD8 + T cells in Mutp53 tumors, whereas CXCR3 inhibition significantly impaired this recruitment. o Quantification of GZMB+CD8 + T cells across treatment groups, confirming that the enrichment induced by PD-1 blockade in Mutp53 tumors was largely abolished by AMG-487 treatment. Note: Data are presented as the mean ± SD. Statistical significance was assessed using one-way or two-way ANOVA with Tukey’s multiple-comparison test, as appropriate. p < 0.05, p < 0.01, * p < 0.001; ns, not significant

Article Snippet: The mouse prostate cancer cell lines RM-1 (CRL-3310TM) and MyC-CaP (CRL-3255TM) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which provides authenticated lines originally sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Protein-Protein interactions, Immunopeptidomics, Immunohistochemical staining, Immunohistochemistry, Western Blot, Chemotaxis Assay, Derivative Assay, Clinical Proteomics, In Vivo, Disruption, Injection, Drug discovery, Multiplex Assay, Immunofluorescence, Staining, Inhibition, Comparison

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mutant p53 epigenetically rewires CXCL10 to promote CD8⁺ T-cell infiltration and enhance the anti-PD-1 response in advanced prostate cancer

doi: 10.1186/s13046-026-03672-z

Figure Lengend Snippet: Mutant p53 occupies the Cxcl10 promoter and remodels promoter-proximal chromatin to enable Cxcl10 transcription. a Immunoblot of RM-1 tumors showing that siRNA-mediated Trp53 silencing reduces the CXCL10 protein. p53 and GAPDH were used as target and loading controls, respectively. b JASPAR motif analysis identifying putative p53-binding sequences within the Cxcl10 promoter. The schematic shows the positions of Primer 1 and Primer 2 relative to the transcription start site and Exon 1. c-d ChIP-qPCR analysis of p53 occupancy at the Cxcl10 promoter. Mutp53 was more strongly enriched than WTp53 at Primer 2, whereas enrichment at Primer 1 was modest. The signals are normalized to the input signal and expressed relative to the IgG signal. e-f Histone-mark ChIP-qPCR analysis of the Cxcl10 promoter. At Primer 1 ( e ), H3K4me3 is minimally enriched in WTp53 tumors but is increased in Mutp53 tumors. At Primer 2 ( f ), Mutp53 tumors display increased H3K4me3 together with reduced H3K27me3 and H3K36me3, which is consistent with a promoter environment permissive for transcription. IgG, negative control. g Agarose-gel electrophoresis of representative ChIP amplicons validating the expected products for the indicated antibodies in WTp53 and Mutp53 tumors; left, bp ladder. h Working model: Mutp53 preferentially occupies the Cxcl10 promoter (primer 2), recruits the activating machinery, increases H3K4me3, and relieves repressive marks (H3K27me3, H3K36me3) relative to WTp53, thereby facilitating RNA polymerase II-driven Cxcl10 transcription. Note: Statistics. The dots represent biologically independent samples (typically n = 3 per group); the bars represent the means ± SEMs. Unless otherwise indicated, ChIP-qPCR datasets in c-f were analyzed via two-way ANOVA followed by multiple comparisons tests. Significance thresholds: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. Abbreviations: WTp53, wild-type p53; Mutp53, mutant p53; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; qPCR, quantitative PCR; IgG, immunoglobulin G control IP; Pol II, RNA polymerase II; TSS, transcription start site; SEM, standard error of the mean; bp, base pairs; kDa, kilodaltons

Article Snippet: The mouse prostate cancer cell lines RM-1 (CRL-3310TM) and MyC-CaP (CRL-3255TM) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which provides authenticated lines originally sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Mutagenesis, Western Blot, Binding Assay, ChIP-qPCR, Negative Control, Agarose Gel Electrophoresis, Small Interfering RNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Control

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: Mouse prostate cancer cells RM1 (Cat# CRL-3310; RRID: CVCL_B459), human prostate cancer cells 22Rv1 (Cat# CRL-2505; RRID: CVCL_1045) and LnCap (Cat# CRL-1740; RRID: CVCL_0395), human monocytic-leukemia cells THP1 (Cat# TIB-202; RRID: CVCL_0006), and mouse fibroblasts L929 (Cat# CCL-1; RRID: CVCL_AR58) were purchased from ATCC and cultured in accordance with the instructions provided by the manufacturer.

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: Mouse prostate cancer cells RM1 (Cat# CRL-3310; RRID: CVCL_B459), human prostate cancer cells 22Rv1 (Cat# CRL-2505; RRID: CVCL_1045) and LnCap (Cat# CRL-1740; RRID: CVCL_0395), human monocytic-leukemia cells THP1 (Cat# TIB-202; RRID: CVCL_0006), and mouse fibroblasts L929 (Cat# CCL-1; RRID: CVCL_AR58) were purchased from ATCC and cultured in accordance with the instructions provided by the manufacturer.

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: Mouse prostate cancer cells RM1 (Cat# CRL-3310; RRID: CVCL_B459), human prostate cancer cells 22Rv1 (Cat# CRL-2505; RRID: CVCL_1045) and LnCap (Cat# CRL-1740; RRID: CVCL_0395), human monocytic-leukemia cells THP1 (Cat# TIB-202; RRID: CVCL_0006), and mouse fibroblasts L929 (Cat# CCL-1; RRID: CVCL_AR58) were purchased from ATCC and cultured in accordance with the instructions provided by the manufacturer.

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: Mouse prostate cancer cells RM1 (Cat# CRL-3310; RRID: CVCL_B459), human prostate cancer cells 22Rv1 (Cat# CRL-2505; RRID: CVCL_1045) and LnCap (Cat# CRL-1740; RRID: CVCL_0395), human monocytic-leukemia cells THP1 (Cat# TIB-202; RRID: CVCL_0006), and mouse fibroblasts L929 (Cat# CCL-1; RRID: CVCL_AR58) were purchased from ATCC and cultured in accordance with the instructions provided by the manufacturer.

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: Mouse prostate cancer cells RM1 (Cat# CRL-3310; RRID: CVCL_B459), human prostate cancer cells 22Rv1 (Cat# CRL-2505; RRID: CVCL_1045) and LnCap (Cat# CRL-1740; RRID: CVCL_0395), human monocytic-leukemia cells THP1 (Cat# TIB-202; RRID: CVCL_0006), and mouse fibroblasts L929 (Cat# CCL-1; RRID: CVCL_AR58) were purchased from ATCC and cultured in accordance with the instructions provided by the manufacturer.

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: Mouse prostate cancer cells RM1 (Cat# CRL-3310; RRID: CVCL_B459), human prostate cancer cells 22Rv1 (Cat# CRL-2505; RRID: CVCL_1045) and LnCap (Cat# CRL-1740; RRID: CVCL_0395), human monocytic-leukemia cells THP1 (Cat# TIB-202; RRID: CVCL_0006), and mouse fibroblasts L929 (Cat# CCL-1; RRID: CVCL_AR58) were purchased from ATCC and cultured in accordance with the instructions provided by the manufacturer.

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: Mouse prostate cancer cells RM1 (Cat# CRL-3310; RRID: CVCL_B459), human prostate cancer cells 22Rv1 (Cat# CRL-2505; RRID: CVCL_1045) and LnCap (Cat# CRL-1740; RRID: CVCL_0395), human monocytic-leukemia cells THP1 (Cat# TIB-202; RRID: CVCL_0006), and mouse fibroblasts L929 (Cat# CCL-1; RRID: CVCL_AR58) were purchased from ATCC and cultured in accordance with the instructions provided by the manufacturer.

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

Journal: Cell reports

Article Title: BCL2 drives castration resistance in castration-sensitive prostate cancer by orchestrating reciprocal crosstalk between oncogenic pathways

doi: 10.1016/j.celrep.2025.115779

Figure Lengend Snippet: (A) Schema showing the experimental design for venetoclax and ENZA combination treatment on castration-sensitive mouse GEM (genetically engineered mouse) tumor-derived cell CSPC (MYCCaP) in immunogenic FVB mice. Schema created with BioRender.com . (B) Growth curve of subcutaneously implanted MYCCaP cells ( n = 8 each group) treated with vehicle control (% DMSO, 30% PEG300), ENZA (30 mg/kg body weight), venetoclax (100 mg/kg body weight), and combination (ENZA, 30 mg/kg; venetoclax, 100 mg/kg). Drugs were delivered to the mice through oral gavage, once each day, until the end of the experiment. Error bar, SD; unpaired t test. (C) Growth curve of individual mouse tumor growth during the combination-treatment study. (D) Survival curve of the mice. Error bar, SD; log rank Mantel-Cox test. Statistical values are calculated by GraphPad Prism. (E) Representative histopathological (scale bar, 500 μm) and immunohistochemical (scale bar, 100 μM) staining of AR and KI67 in the MYCCaP mouse tumors. (F) The graph represents the number of ki67-positive cells/high power field (hpf) in the mouse tumors of each group. 10 hpf of each tumor sections were selected. Error bar, SD; unpaired t test. (G) Western blot showing the expression of indicated proteins in MYCCaP tumors either treated with vehicle or ENZA. Each lane represents an individual mouse tumor. (H) LNCaP cells were transiently transfected with BCL2 siRNA or scrambled/control (Scr) siRNA. 48 h post transfection, cells (1 × 10 4 /well) were cultured in a low-adherence plate for anchorage-independent tumorsphere-formation assay in tumorsphere medium supplemented with 1 nM R1881 alone or in combination with 10 μM ENZA (ENZ) for another 2 weeks. Equivalent volume of DMSO was used as placebo treatment. The representative images (50× magnification) showing the changes of tumorsphere formation in response to BCL2 knockdown and ENZ treatment. Scale bar, 500 μm. (I) WB showing the expression of indicated proteins in LNCaP-abl cells treated with 0.1 and 1 μM MK2206 for 24 h under ADT (CSS). n = 3 biological replicates. (J) qPCR showing the expression of BCL2 mRNA expression in LNCaP-Abl cells when treated with 0.1 and 1 μM MK2206 (Akt inhibitor) for 24 h under ADT (CSS). n = 3 biological replicates. Error bar, SD. (K) Schema of the regulation of BCl2 expression in castration-resistant PTEN-null PC cells. (L) Bar graph showing the cell growth of LNCaP-Abl cells when treated with venetoclax (7.7 μM), MK2206 (1 μM), or in combination for 7 days. Cell growth was measured by MTT. Error bar, SD; unpaired t test. Data represent eight biological replicates.

Article Snippet: Human prostate cancer cells LNCaP, 22RV1, DU145, PC3, VCaP, human breast carcinoma cells MDA-MB-231 and castration sensitive mice prostate cancer cell MYCCaP were obtained from ATCC (Manassas, VA).

Techniques: Derivative Assay, Control, Immunohistochemical staining, Staining, Western Blot, Expressing, Transfection, Cell Culture, Tube Formation Assay, Knockdown