Journal: Journal for Immunotherapy of Cancer

Article Title: Inhibition of stromal MAOA leading activation of WNT5A enhance prostate cancer immunotherapy by involving the transition of cancer-associated fibroblasts

doi: 10.1136/jitc-2024-010555

Figure Lengend Snippet: Suppressing stromal MAOA activity in vivo can effectively inhibit the proliferation of prostate cancer xenografts by potentiating the immune-killing function of CD8 + T cells. ( A ) Schematic diagram of the construction of a subcutaneous tumor graft model in C57BL/6J mice and the drug administration strategy. ( B ) Tumor growth curves of mice in different treatment groups. ( C ) Representative images and quantification results of KI-67 + and Tunel + in mice from different treatment groups, scale bars, 50 µm. ( D ) Representative images and quantification results of CD8 + T cells in different treatment groups of mice, scale bars, 50 µm. ( E ) FACS analysis of the proportion of CD8 + CD45 + T cells in tumor tissues from different treatment groups. ( F ) ELISA experiments to detect the content of IFN-γ in tumor tissues from different treatment groups. ( G ) Strategy for the construction of double humanized PCa mouse model and administration. ( H ) The tumor growth curves of mice in the NC group and the sh-MAOA group. ( I ) Representative images and quantification results of KI-67 + and Tunnel + for mice in the NC group and sh-MAOA group, scale bars, 50 µm. ( J ) Representative images and quantification results of CD8 + T cells in the NC group and sh-MAOA group of mice. Scale bars, 50 µm. ( K ) FACS analysis of the proportion of CD8 + CD45 + T cells in tumor tissues from NC group and sh-MAOA group. ( L ) ELISA experiments to detect the content of IFN-γ in tumor tissues from NC group and sh-MAOA group. Clo, chlorgyline; Con, control; DAPI, 4',6-Diamidino-2-Phenylindole; FACS, fluorescence-activated cell sorting; M-PrSC, mouse primary stromal cells; NC, negative control; PBMC, peripheral blood mononuclear cell; PCa, prostate cancer; Phe, phenelzine; sh-MAOA, short hairpin RNA targeting monoamine oxidase A.

Article Snippet: Human PCa cell lines (PC3, DU145, C42, 22RV1), mouse PCa cell lines (RM-1, Tramp-C1), Human prostate stromal myofibroblast (WPMY-1) and Human leukemia cell line (JURKAT) were obtained from the American Type Culture Collection (Manassas, Virginia, USA) and cultured in Roswell Park Memorial Institute 1640 and Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (Gibco).

Techniques: Activity Assay, In Vivo, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Control, Fluorescence, FACS, Negative Control, shRNA

Journal: Journal for Immunotherapy of Cancer

Article Title: Inhibition of stromal MAOA leading activation of WNT5A enhance prostate cancer immunotherapy by involving the transition of cancer-associated fibroblasts

doi: 10.1136/jitc-2024-010555

Figure Lengend Snippet: Inhibition of stromal MAOA sensitizes prostate cancer cells to the therapeutic effects of immune checkpoint inhibitors. ( A ) Strategy for the construction of subcutaneous tumor xenograft model in C57BL/6J mouse model and administration. ( B ) Tumor growth curves of mice in different treatment groups. ( C ) The expression of WNT5A, the proportion of CD8 + T cells, and the content of IFN-γ in different treatment groups of mice tumor tissues. ( D ) Strategy for the construction of double humanized PCa mouse model and administration. ( E ) Tumor growth curves of mice in different treatment groups. ( F ) The expression of WNT5A, the proportion of CD8 + T cells, and the content of IFN-γ in different treatment groups of mice tumor tissues. ( G ) A schematic diagram of the mechanism by which inhibiting MAOA in tumor-associated fibroblasts exerts antitumor effects. Availability of data and material: the datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. CAF, cancer-associated fibroblast; Clo, chlorgyline; Con, Control; MAOA, monoamine oxidase A; M-PrSC, mouse primary stromal cells; ns, not significant; PBMC, peripheral blood mononuclear cell; PCa, prostate cancer; PD-1, programmed cell death protein-1; ROS, Reactive Oxygen Species; sh-MAOA, short hairpin RNA targeting MAOA; WT, Wild Type.

Article Snippet: Human PCa cell lines (PC3, DU145, C42, 22RV1), mouse PCa cell lines (RM-1, Tramp-C1), Human prostate stromal myofibroblast (WPMY-1) and Human leukemia cell line (JURKAT) were obtained from the American Type Culture Collection (Manassas, Virginia, USA) and cultured in Roswell Park Memorial Institute 1640 and Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (Gibco).

Techniques: Inhibition, Expressing, Generated, Control, shRNA

Journal: EMBO Reports

Article Title: Prostate cancer cell‐intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone

doi: 10.15252/embr.202050162

Figure Lengend Snippet: PKH26 (PE)‐labeled RM1 (V500/eCFP) cells were injected intracardiacally (IC) into C57BL/6 mice and FACS‐isolated from bones with evident tumor burden (eight individual mice across five independent experiments) at ∼ day 16, and individual dormant and proliferating cells were isolated for scRNA‐seq. Representative tumor burden at the whole mouse level and in bone shown by bioluminescence with representative FACS plots of PKH + (PE/V500) and PKH − (V500) RM1 cells and bone marrow cells (gray). goana gene ontology (GO) analysis (limma) of all DE genes enriched in proliferating (PKH − , n = 32) cells compared to dormant cells (PKH + , n = 28). Gene sets appear in order of significance ( P ‐value) with color representing fold enrichment and bar width indicating the number of genes in each process. goana GO analysis (limma) of all DE genes uniquely enriched in PKH + compared to PKH − cells. Gene sets appear in order of significance ( P ‐value) with color representing fold enrichment and bar width indicating the number of genes in each process. INTERFEROME database classification of differentially expressed (DE) genes retained in dormant cells into predicted type I and/or II IFN‐regulated genes (IRGs). Heatmap of relative BASiCS‐derived log 2 (denoised counts + 1) of type I IRGs (upregulated > twofold in the INTERFEROME in at least one dataset) that are differentially expressed between all dormant and active cells with no residual overdispersion. Mouse IDs for each sample are indicated, along with type I IRGs that are identified as such in at least 30 INTERFEROME datasets. Genes are displayed if detected in at least 10 samples per population. Samples with zero counts for individual genes are represented in white. Dot plot of BASiCS‐derived log 2 (denoised counts + 1) of key DE IRGs expressed in > 5 dormant cells associated with immune‐activatory processes (among one of the most enriched types of biological pathway determined by GO analyses) present for all single cells ranked by ExpLogFC (Fig C). Open circles are zero counts. Bars indicate mean expression. Source data are available online for this figure.

Article Snippet: To assess the efficacy of HDACi and poly I:C on bone metastasis inhibition in RM1 BD Irf − tumor‐bearing mice, the following groups ( n = 6) were administered: vehicle (1 × dose of saline; 1 × dose of DSPT [2% DMSO, 0.09% saline, 30% PEG 300, and 2% Tween‐80]); MS275 (8 mg/kg in DSPT; 1 × dose saline; Selleckchem); poly I:C (25 mg/mouse in saline; 1 × dose DSPT); and Combo (1 × dose MS275; 1 × dose poly I:C).

Techniques: Labeling, Injection, Isolation, Derivative Assay, Expressing

Journal: EMBO Reports

Article Title: Prostate cancer cell‐intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone

doi: 10.15252/embr.202050162

Figure Lengend Snippet: A–C Bulk RNA‐seq analysis of DE genes (DEG) significantly suppressed (FDR < 0.05; generalized linear model extraction (GLM) by edgeR) in proliferating RM1 bone‐derived (BD) cells compared to parental RM1 and lung metastases. Tumor‐intrinsic type I IFN signaling is suppressed in proliferating bone lesions ( n = 1) compared to parental RM1 ( n = 2) cells as shown by (A) camera analysis of Hallmark gene set responses associated with bone metastasis (bar length is the –log 10 FDR, false discovery rate). Color indicates mean log 2 fold change (FC) of all genes in gene set. Bar width is relative gene set size. Dashed line shows FDR = 0.05; (B) barcode plot showing enrichment of the Hallmark IFN‐α response gene set in genes suppressed in bone metastases, using the signed log‐likelihood ratio statistic (EdgeR). Bars indicate value of the statistic for each gene in the gene set; (C) log 2 FC values of key downregulated IRGs, with genes retained in dormant cells and classical downstream IFN signaling targets indicated. Altered IRGs directly involved in IFN‐α/β production (light blue) compared to downstream IFN targets (dark blue) are segregated. P ‐values represented as * < 0.05, ** < .005, and *** < 0.0005; GLM by edgeR. D Schematic of proliferating RM1 cell isolation from bone and lung metastases following IC injection for preliminary RNA‐seq and orthogonal validation by qRT–PCR. E Heatmap of mean normalized voom expression of IRGs suppressed in RM1 bone metastases ( n = 1) compared to lung metastases ( n = 1) and parental cells ( n = 2). F qRT–PCR validation of Irf7 and Irf9 downregulation in RM1 cells from bone metastases (RMI BD) compared to parental RM1 cells, lung metastases (RM1 lung), and naïve bone marrow (BM) ( n = 3 mice per group). P ‐values represented as ** < 0.005, and *** < 0.0005 (Student's t ‐test). G qRT–PCR of Irf7 and Irf9 downregulation in parental RM1 cells and RM1 cells from bone metastases (RM1 BD) in WT and Ifnar1 ‐deficient (−/−) mice, with naïve BM from WT and Ifnar −/− animals for reference. ( n = 3 mice per group, n = 1 for Ifnar −/− BM control). P ‐values were represented as * < 0.05 and ** < 0.005 (Student's t ‐test). Data information: All error bars ± SEM. Source data are available online for this figure.

Article Snippet: To assess the efficacy of HDACi and poly I:C on bone metastasis inhibition in RM1 BD Irf − tumor‐bearing mice, the following groups ( n = 6) were administered: vehicle (1 × dose of saline; 1 × dose of DSPT [2% DMSO, 0.09% saline, 30% PEG 300, and 2% Tween‐80]); MS275 (8 mg/kg in DSPT; 1 × dose saline; Selleckchem); poly I:C (25 mg/mouse in saline; 1 × dose DSPT); and Combo (1 × dose MS275; 1 × dose poly I:C).

Techniques: RNA Sequencing, Extraction, Derivative Assay, Cell Isolation, Injection, Biomarker Discovery, Quantitative RT-PCR, Expressing, Control

Journal: EMBO Reports

Article Title: Prostate cancer cell‐intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone

doi: 10.15252/embr.202050162

Figure Lengend Snippet: Stability of Irf7 and Irf9 mRNA suppression by qRT–PCR in ex vivo bone‐derived cells (RM1 BD Irf − , n = 7) in culture compared to a bone‐derived line that showed initial loss during early passage (RM1 BD REV (EP), n = 3), then reverted to parental expression levels at late passage (RM1 BD REV (LP), n = 4) compared to parental RM1 ( n = 3). ELISA of IFN‐α production by RM1 parental ( n = 3), RM1 bone‐derived Irf‐low (RM1 BD Irf − , n = 4), and RM1 BD REV ( n = 3) cells subsequent to poly I:C stimulation. qRT–PCR analysis of Irf7 and Irf9 expression in RM1 BD Irf − cells ± 48 h treatment with MS275 (1 μM) ( n = 7–9). Schematic of contact and transwell co‐culture systems. qRT–PCR analysis of Irf7 and Irf9 expression in parental RM1 cells ( n = 4) 48 h post‐contact culture with naïve BM ( n = 6). qRT–PCR analysis of Irf9 expression in parental RM1 cells ± 48 h co‐culture with naïve BM under contact (non‐transwell; NT) and transwell (0.4‐μm filters that prevent cell contact) conditions ( n = 6–8 per condition). qRT–PCR analysis of Irf9 expression in parental RM1 cells ± 48 h contact co‐culture with naïve BM ± MS275 (1 μM) ( n = 3–6 per condition). Data information: P ‐values represented as * < 0.05, ** < 0.005, and *** < 0.0005 (Student's t ‐test). All error bars ± SEM.

Article Snippet: To assess the efficacy of HDACi and poly I:C on bone metastasis inhibition in RM1 BD Irf − tumor‐bearing mice, the following groups ( n = 6) were administered: vehicle (1 × dose of saline; 1 × dose of DSPT [2% DMSO, 0.09% saline, 30% PEG 300, and 2% Tween‐80]); MS275 (8 mg/kg in DSPT; 1 × dose saline; Selleckchem); poly I:C (25 mg/mouse in saline; 1 × dose DSPT); and Combo (1 × dose MS275; 1 × dose poly I:C).

Techniques: Quantitative RT-PCR, Ex Vivo, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

Journal: EMBO Reports

Article Title: Prostate cancer cell‐intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone

doi: 10.15252/embr.202050162

Figure Lengend Snippet: Assessment of tumor‐intrinsic IFN suppression stability over passage (P; number indicated) in culture by qRT–PCR analysis of mean Irf7 and Irf9 mRNA expression in bone‐derived RM1 Irf‐low (RM1 BD Irf − ) cells and a reverted (REV) bone‐derived cell line compared to RM1 parental cells. Values are means ± SEM of three independent experiments. HDACi impact on RM1 BD Irf − proliferation over time by SRB assay. Mean OD at 550 nm ( n = 3). Flow cytometry characterization of bone marrow lymphocyte (%) populations ( n = 3). qRT–PCR of Irf9 expression in parental RM1 cells 48, 72, and 96 h post‐contact co‐culture with FACS‐isolated naïve CD11b + Ly6G + BM cells ( n = 3 mice per time point). qRT–PCR of Irf7 expression in RM1 parental cells ± co‐culture with naïve BM ± 48 h treatment with MS275 ( n = 3–6). P ‐values represented as * < 0.05, ** < 0.005, and *** < 0.0005 (Student's t‐ test). All error bars ± SEM.

Article Snippet: To assess the efficacy of HDACi and poly I:C on bone metastasis inhibition in RM1 BD Irf − tumor‐bearing mice, the following groups ( n = 6) were administered: vehicle (1 × dose of saline; 1 × dose of DSPT [2% DMSO, 0.09% saline, 30% PEG 300, and 2% Tween‐80]); MS275 (8 mg/kg in DSPT; 1 × dose saline; Selleckchem); poly I:C (25 mg/mouse in saline; 1 × dose DSPT); and Combo (1 × dose MS275; 1 × dose poly I:C).

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Sulforhodamine B Assay, Flow Cytometry, Co-Culture Assay, Isolation

Journal: EMBO Reports

Article Title: Prostate cancer cell‐intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone

doi: 10.15252/embr.202050162

Figure Lengend Snippet: A Bone metastasis‐free survival in WT C57BL/6 mice harboring RM1 parental, RM1 BD Irf − , and RM1 BD REV (LP) tumors ( n = 5 per group; ** P = 0.0043 by log‐rank (Mantel–Cox) test). B Schematic of enforced Irf7 expression in RM1 BD Irf − cells under exogenous promoter control. C ELISA of IFN‐α production by RM1 parental, RM1 BD Irf − base vector (BV), and RM1 BD Irf7 overexpressing (OE) with 24‐h poly I:C stimulation ( n = 4). D Bone metastasis‐free survival in WT C57BL/6 mice subsequent to IC inoculation of RM1 BD Irf − BV ( n = 9) and RM1 BD Irf7 OE ( n = 7) cells (** P = 0.0028 by log‐rank (Mantel–Cox) test). E Bone metastasis‐free survival in Ifnar1 −/− mice harboring RM1 BD Irf − BV and RM1 BD Irf7 OE tumors ( n = 4 per group) and representative bioluminescent imaging of tumor burden in leg bones (also shown in Appendix Fig S4F ). F Immunohistochemical (IHC) staining for cerulean (anti‐eCFP; green) on naïve WT bone, RM1 parental, RM1 BD Irf − BV, and RM1 BD Irf7 OE tumor‐bearing bones derived from WT animals; and RM1 BD Irf7 OE tumor‐bearing bones derived from Ifnar1 −/− animals at survival assay endpoints. Blue represents DAPI nuclear staining. Scale bar, 100 μm. G–I FACS analysis of dormant (claret + ) RM1 BD Irf − and RM1 BD Irf7 OE cells from bone at (G) day 11 post‐IC injection ( n = 4 per group) and (H) day 17 ( n = 4–6 mice group/time point) with (I) D17 active (claret − ) colony quantitation (mean +1) determined by multiphoton imaging and IMARIS interrogation ( n = 9 bones from three mice per condition). Median shown. Upper and low box hinges denote first and third quartiles. Whiskers mark value limits. J FACS analysis of IFN‐γ + and TNF‐α + CD8 + T cells (%) post‐ICS induction of T‐cell (spleen derived from tumor‐bearing mice) activation by RM1 parental, RM1 BD Irf − BV, and RM1 BD Irf7 OE cells ( n = 6 per condition/group). K Mean quantitation of TRAP‐stained osteoclasts differentiated with M‐CSF ± RANKL in co‐culture with RM1 BD Irf − BV and RM1 BD Irf7 OE cells with representative wells shown ( n = 3 per condition M‐CSF; n = 6 per condition M‐CSF + RANKL; also shown in Appendix Fig S4J ). Scale bar represents 100 μm. Data information: P‐ values represented as * < 0.05, ** < 0.005, and *** < 0.0005 (Student's t ‐test). All error bars ± SEM, except where stated.

Article Snippet: To assess the efficacy of HDACi and poly I:C on bone metastasis inhibition in RM1 BD Irf − tumor‐bearing mice, the following groups ( n = 6) were administered: vehicle (1 × dose of saline; 1 × dose of DSPT [2% DMSO, 0.09% saline, 30% PEG 300, and 2% Tween‐80]); MS275 (8 mg/kg in DSPT; 1 × dose saline; Selleckchem); poly I:C (25 mg/mouse in saline; 1 × dose DSPT); and Combo (1 × dose MS275; 1 × dose poly I:C).

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Imaging, Immunohistochemical staining, Immunohistochemistry, Derivative Assay, Clonogenic Cell Survival Assay, Staining, Injection, Quantitation Assay, Activation Assay, Co-Culture Assay

Journal: EMBO Reports

Article Title: Prostate cancer cell‐intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone

doi: 10.15252/embr.202050162

Figure Lengend Snippet: Representative H&E‐ and TRAP‐stained bone sections from naïve and RM1 BD Irf − tumor‐bearing animals. T indicates tumor regions. Scale bar, 200 μm.

Article Snippet: To assess the efficacy of HDACi and poly I:C on bone metastasis inhibition in RM1 BD Irf − tumor‐bearing mice, the following groups ( n = 6) were administered: vehicle (1 × dose of saline; 1 × dose of DSPT [2% DMSO, 0.09% saline, 30% PEG 300, and 2% Tween‐80]); MS275 (8 mg/kg in DSPT; 1 × dose saline; Selleckchem); poly I:C (25 mg/mouse in saline; 1 × dose DSPT); and Combo (1 × dose MS275; 1 × dose poly I:C).

Techniques: Staining

Journal: EMBO Reports

Article Title: Prostate cancer cell‐intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone

doi: 10.15252/embr.202050162

Figure Lengend Snippet: A ELISA of IFN‐α production by RM1 BD Irf − cells ± single‐agent MS275 and poly I:C treatment or 48 h pre‐treatment with MS275 (pMS) prior to poly I:C stimulation ( n = 6). B Mean fluorescence intensity of H2‐Kb staining on RM1 BD Irf − cells by FACS ± MS275, poly I:C, or combination treatment ( n = 3). C, D FACS analysis of IFN‐γ + CD8 + T cells (%) post‐ICS induction of T‐cell (spleen derived from RM1 BD Irf − tumor‐bearing mice) activation upon re‐stimulation with RM1 cells and MS275, poly I:C, or combination treatment, with NAC (no antigen‐presenting cells) and RM1 BD Irf7 OE controls ( n = 3; Irf7 OE control (C), n = 1) with (D) representative FACS plots shown. E Bone metastasis‐free survival in WT C57BL/6 mice harboring RM1 BD Irf − cells ± MS275, poly I:C, or combination treatment ( n = 6 per group; log‐rank (Mantel–Cox) test). F Mean total tumor burden in bone (femurs, tibias, spine, and humerus) at endpoint by bioluminescent intensity score (log 2 p/s/cm 2 /sr) with values < 4.0 × 10 4 representing zero burden ( n = 24 bones per group; six mice per group) and representative bioluminescent imaging of tumor burden in spine (all shown in Appendix Fig S5E ). Data information: P ‐values represented as * < 0.05, ** < 0.005, and *** < 0.0005 (Student's t ‐test in A–C and F; log‐rank [Mantel–Cox] test in E). All error bars ± SEM.

Article Snippet: To assess the efficacy of HDACi and poly I:C on bone metastasis inhibition in RM1 BD Irf − tumor‐bearing mice, the following groups ( n = 6) were administered: vehicle (1 × dose of saline; 1 × dose of DSPT [2% DMSO, 0.09% saline, 30% PEG 300, and 2% Tween‐80]); MS275 (8 mg/kg in DSPT; 1 × dose saline; Selleckchem); poly I:C (25 mg/mouse in saline; 1 × dose DSPT); and Combo (1 × dose MS275; 1 × dose poly I:C).

Techniques: Enzyme-linked Immunosorbent Assay, Fluorescence, Staining, Derivative Assay, Activation Assay, Control, Imaging

Journal: EMBO Reports

Article Title: Prostate cancer cell‐intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone

doi: 10.15252/embr.202050162

Figure Lengend Snippet: A, B FACS analysis of (A) peripheral blood (PB) T and NK lymphocyte representation and activation status and (B) FoxP3 + CD4 + and CD4 + effector memory T‐cell status at days 4 and 10 post‐IC tumor cell inoculation across treatment settings ( n = 4–6 per group). C, D Bioluminescent imaging (C) of a combination group mouse with tumor clearance from days 7 to 41 (full experimental cohort shown in Appendix Fig S5E ) post‐IC inoculation with (D) associated specific CD8 + memory T‐cell response. This is represented by FACS analysis of IFN‐γ + CD8 + T cells (%) post‐ICS induction of T‐cell (spleen derived from tumor‐bearing mice) activation upon re‐stimulation with RM1 BD Irf − cells and MS275, poly I:C, or combination treatments in vitro , with NAC and RM1 BD Irf7 OE controls ( n = 3; Irf7 OE control (C), n = 1). Representative FACS plots shown on right. Data information: P ‐values represented as * < 0.05, ** < 0.005, and *** < 0.0005 (Student's t‐ test). All error bars ± SEM.

Article Snippet: To assess the efficacy of HDACi and poly I:C on bone metastasis inhibition in RM1 BD Irf − tumor‐bearing mice, the following groups ( n = 6) were administered: vehicle (1 × dose of saline; 1 × dose of DSPT [2% DMSO, 0.09% saline, 30% PEG 300, and 2% Tween‐80]); MS275 (8 mg/kg in DSPT; 1 × dose saline; Selleckchem); poly I:C (25 mg/mouse in saline; 1 × dose DSPT); and Combo (1 × dose MS275; 1 × dose poly I:C).

Techniques: Activation Assay, Imaging, Derivative Assay, In Vitro, Control