Journal: The Journal of Clinical Investigation

Article Title: PARP inhibitors restore NK cell function via secretory crosstalk with tumor cells in prostate cancer

doi: 10.1172/JCI197157

Figure Lengend Snippet: ( A ) Expression of CD16, CD56, NCR1, and KLRF1 in PCa versus normal tissues (TCGA). The box-and-whisker plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. ( B ) Immunohistochemical staining of NCR1 in high- and low-grade PCa tissues collected at Tongji Hospital. Scale bars: 100 μm. ( C ) Proportion of NCR1 expression in patients with high-grade ( N = 10) versus intermediate/low-grade ( N = 10) PCa. ( D ) Treatment regimens for C57BL/6 mice bearing CRPC-PPSM starting on day 8 after inoculation: enzalutamide (10 mg/kg, oral, daily), anti–PD-1 (200 μg/kg, i.p., every 3 days), docetaxel (10 mg/kg, i.v., weekly), and mefuparib (40 mg/kg, oral, every 2 days). Mice were euthanized on day 25 after tumor inoculation. ( E ) Tumor growth curves and tumor weights in mice from D for each treatment; N = 5 per group. Scale bars: 1 cm. ( F ) Immunofluorescence of tumors from D showing DAPI (blue), NK1.1 (red), GZMB (yellow), and PanCK (white). Scale bars: 20 μm. ( G ) Tumor growth curves and weights of C57BL/6 mice inoculated with RM-1 cells and treated with or without PARPi; N = 6 per group. Scale bars: 1 cm. ( H ) Tumor growth curves and weights of Rag1 – / – γc – / – mice inoculated with RM-1 or RM-1 BRCA1 -KO cells and treated with or without PARPi; N = 5 per group. Scale bars: 1 cm.( I – L ) Flow cytometry analysis of NK cell proportions in tumors ( I ) and peripheral blood ( K ), GZMB + proportion in TINKs ( J ), and TNF-α + IFN-γ + proportions in PBNKs ( L ) of mice from G ; N = 5 per group. Tumor growth curve data are presented as mean ± SD and were analyzed by 2-way ANOVA with Tukey’s multiple-comparison test. Other data are presented as mean ± SEM. Data were analyzed by 1-way ANOVA ( E and H ) and Welch’s t test ( G and I – L ).

Article Snippet: The PARPi treatment used mefuparib (MedChemExpress), which was administered by gavage at a dose of 40 mg/kg per mouse (once every 2 days).

Techniques: Expressing, Whisker Assay, Immunohistochemical staining, Staining, Immunofluorescence, Flow Cytometry, Comparison

Journal: The Journal of Clinical Investigation

Article Title: PARP inhibitors restore NK cell function via secretory crosstalk with tumor cells in prostate cancer

doi: 10.1172/JCI197157

Figure Lengend Snippet: ( A ) Paired peripheral blood serum samples from 5 PCa patients before and after PARPi treatment were analyzed using proteomics profiling. ( B ) Heatmap showing changes in serum protein profiles before and after PARPi treatment. ( C and D ) RT-qPCR analysis of PARPi-induced changes in CypA mRNA levels in murine PCa cell lines ( C ) and human PCa cell lines ( D ); N = 3 per group. ( E and F ) Western blot analysis of PARPi-induced changes in intracellular CypA (cCypA) and eCypA protein levels in murine ( E ) and human ( F ) PCa cell lines. ( G and H ) ELISA of PARPi-induced changes in CypA supernatant levels in murine ( G ) and human ( H ) PCa cell lines. ( I and J ) DCFH-DA (green) staining to detect PARPi-induced ROS levels in PCa cell lines RM-1 ( I ; N = 3) and MycCap/DU145/22RV1 ( J ; N = 5). Original magnification, ×100. ( K ) Gene Ontology enrichment analysis showing enhanced intracellular transport pathways in PCa cells after PARPi stimulation. ( L ) Western blot analysis of cCypA and eCypA levels after protein transport inhibition by Brefeldin A (BFA). Data are presented as mean ± SEM and were analyzed by Welch’s t test.

Article Snippet: The PARPi treatment used mefuparib (MedChemExpress), which was administered by gavage at a dose of 40 mg/kg per mouse (once every 2 days).

Techniques: Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Inhibition

Journal: The Journal of Clinical Investigation

Article Title: PARP inhibitors restore NK cell function via secretory crosstalk with tumor cells in prostate cancer

doi: 10.1172/JCI197157

Figure Lengend Snippet: ( A ) Tumor growth curves and weights in PARPi-treated tumor-bearing mice coadministered with CypA inhibitor CsA and FPR1 inhibitors CsH and HCH6-1; N = 6 per group; scale bars: 1 cm ( B ) Tumor growth curves and weights in C57BL/6 mice bearing RM-1 tumors treated with FPR1 agonist fMIFL or inhibitor HCH6-1; N = 5 per group; scale bars: 1 cm ( C and D ) IFN-γ and GZMB expression levels in TINKs ( C ) and their quantification ( D ) under HCH6-1 or fMIFL treatment; N = 5 per group. ( E ) Western blot analysis of ERK- and AKT-related pathway activation in NK cells treated with CypA, CsA, CsH, or HCH6-1 in vitro. ( F and G ) Flow cytometry analysis of IFN-γ and GZMB expression ( F ) and quantification ( G ) in NK cells treated with FPR1 inhibitor CsH, AKT inhibitor MK-2206, or ERK inhibitor SCH772984; N = 5 per group. ( H ) Transmission electron microscopy images showing mitochondrial structure and crista alterations in NK cells stimulated with CypA. Scale bars: 1 μm. ( I ) Statistical analysis of mitochondrial number ( N = 10) and crista count ( N = 15) in NK cells. Tumor growth curve data are presented as mean ± SD and were analyzed by 2-way ANOVA with Tukey’s multiple-comparison test. Other data are presented as mean ± SEM and were analyzed by 1-way ANOVA ( A , B , D , and G ) and Welch’s t test ( I ).

Article Snippet: The PARPi treatment used mefuparib (MedChemExpress), which was administered by gavage at a dose of 40 mg/kg per mouse (once every 2 days).

Techniques: Expressing, Western Blot, Activation Assay, In Vitro, Flow Cytometry, Transmission Assay, Electron Microscopy, Comparison

Journal: The Journal of Clinical Investigation

Article Title: PARP inhibitors restore NK cell function via secretory crosstalk with tumor cells in prostate cancer

doi: 10.1172/JCI197157

Figure Lengend Snippet: ( A ) Mice bearing RM-1 tumors were treated with mefuparib and adoptive transfer of 10 5 NK cells every 2 days from day 8. Mice were euthanized on day 18 for tumor collection. ( B and C ) Tumor growth curves ( B ) and weights ( C ) in mice treated with adoptive NK cell therapy, PARPi, or combination therapy; N = 6; scale bars: 1 cm. ( D ) Immunofluorescence of tumors from mice treated with adoptive NK cells, PARPi, or combination therapy, showing DAPI (blue), NK1.1 (red), GZMB (yellow), and PanCK (white). Scale bars: 50 μm. ( E and F ) Quantification of average NK1.1 + cells ( E ) and GZMB + NK1.1 + cells ( F ) per high-power field. Data are based on average counts from 5 random fields per sample; N = 5. ( G ) Mice bearing orthotopic PPSM tumors received mefuparib and 10 5 NK cells every 2 days from day 8. Mice were euthanized on day 18 for analysis or retained for survival observation. ( H ) In vivo small animal imaging of orthotopic prostate tumor growth on day 18. ( I ) Survival curves of orthotopic PCa mouse models. ( J ) Western blot confirming hPSMA expression in DU145 cells. ( K ) Design of CAR-NK cells targeting human PSMA. SP, signal peptide; TM, transmembrane. ( L ) Representative flow cytometry analysis showing the transduction efficiency of CAR-NK cells. ( M ) Establishment of the DU145-hPSMA in situ PCa implantation model. Subsequently, tumor-bearing mice were treated with mefuparib and 10 5 CAR-NK cells every 2 days from day 8. Mice were euthanized on day 24 for tumor collection. ( N ) Tumor weight in DU145-hPSMA–bearing mice treated with PARPi and/or CAR-NK cells; N = 6. Tumor growth curve data are presented as mean ± SD and were analyzed by 2-way ANOVA with Tukey’s multiple-comparison test. Survival curves were analyzed by a log-rank (Mantel-Cox) test. Other data are presented as mean ± SEM and were analyzed by 1-way ANOVA.

Article Snippet: The PARPi treatment used mefuparib (MedChemExpress), which was administered by gavage at a dose of 40 mg/kg per mouse (once every 2 days).

Techniques: Adoptive Transfer Assay, Immunofluorescence, In Vivo, Imaging, Western Blot, Expressing, Flow Cytometry, Transduction, In Situ, Comparison