Journal: Neurourology and urodynamics

Article Title: A possible mechanism underlying mood disorders associated with LUTS: Chronic bladder outlet obstruction causes NLRP3-dependent inflammation in the hippocampus and depressive behavior in rats

doi: 10.1002/nau.24448

Figure Lengend Snippet: After 12 weeks of BOO, neurogenesis is statistically decreased in the hippocampus and this increase is blocked by glyburide treatment. Cells in 10 μm sections of brain were stained for Ki-67 and then visualized and quantitated as described in the Materials and Method section. The results are presented as the density of Ki-67+ cells per μm2. Results are the mean ± 95% confidence intervals; *P < .05, ***P < .005 by ANOVA and Dunn’s test (n = 6, 6, 6, and 6). ANOVA, analysis of variance; BOO, bladder outlet obstruction; Gly, glyburide-treated; Veh, vehicle-treated

Article Snippet: Coronal sections (10 μm) were stained with anti-IbA1/AIF1 (1:500) (NBP2-19019; Novus Biologicals, Centennial, CO) or anti-Ki67 (1:500) (ab15580; Abcam, Cambridge, MA) using standard methodology and citrate antigen retrieval.

Techniques: Staining

Journal: Journal for immunotherapy of cancer

Article Title: Understanding the dynamics of TKI-induced changes in the tumor immune microenvironment for improved therapeutic effect.

doi: 10.1136/jitc-2024-009165

Figure Lengend Snippet: Figure 1 TKI therapy alters the tumor immune microenvironment. (A) Representative mIF images of pretreatment tumor and resected samples analyzed for immune-related biomarkers. (B) Densities (cells/mm2) of CD8+, CD8+GB+, CD8+PD-1+, CD163+, CD68+, and CD163+CD68+ by mIF quantification in paired pretreatment tumor samples and resected tumors. (C) Cell viability CCK-8 assay for cells treated with TKIs (osimertinib: 10 nM, lorlatinib: 10 nM), activated T cells (1:1 ratio to cancer cells), or the combination. (D) T cell-mediated cancer cell-killing assay. PC-9 and H3122 cells co-cultured with activated T cells for 48 hours with or without TKIs (osimertinib: 10 nM, lorlatinib: 10 nM) were subjected to crystal violet staining. Ratio of cancer cells to T cells: 1:1. (E) Ki67 incorporation assay on PC-9 and H3122 cells treated as indicated. Activated T cells (1:1 ratio to cancer cells) or TKIs (osimertinib: 10 nM, lorlatinib: 10 nM) were added to the culture medium for 48 hours. Cells were then counterstained with DAPI. (F) PC-9 cells were injected into mice (n=3 mice per group) on day 0, hu-PBMCs were injected into mice via the tail vein on day 7, and osimertinib was administered as indicated. (G) Macroscopic appearance of tumors after drug application for 4 weeks. (H) The tumor weight (g) for each mouse is shown. *p<0.05, **p<0.01. ns, no significance. (I) Immunofluorescence staining with an antibody against CD8 to detect T cells and antibodies against CD68 and CD206 to detect macrophages in TKI-resistant non-small cell lung cancer tissues (11 cases of EGFR-TKI resistance, 5 cases of ALK-TKI resistance). Scale bar: 50 µm. ALK, anaplastic lymphoma kinase; DAPI, 4′,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; ALKi,anaplastic lymphoma kinase inhibitor; EGFRi,epidermal growth factor receptor inhibitor; hu-PBMC,human-Peripheral blood mononuclear cell; mIF, multiplex immunofluorescence; PD-1, programmed cell death protein 1; TKI, tyrosine kinase inhibitor.

Article Snippet: Then, the cells were fixed and incubated overnight with Ki67 (#M00254- 8, Boster, Wuhan, China).

Techniques: CCK-8 Assay, Cell Culture, Staining, Injection, Immunofluorescence, Multiplex Assay

Journal: Journal for immunotherapy of cancer

Article Title: Understanding the dynamics of TKI-induced changes in the tumor immune microenvironment for improved therapeutic effect.

doi: 10.1136/jitc-2024-009165

Figure Lengend Snippet: Figure 6 Aspirin enhances the antitumor immunity response. (A) PC-9 cells were injected into mice (n=3 mice per group) on day 0, hu-PBMCs were injected into the tail veins of mice on day 7, and osimertinib/osimertinib plus aspirin was administered as indicated. (B) Macroscopic appearance of tumors after drug application for 4 weeks. The tumor weight (g) of each mouse is shown. *p<0.05, ***p<0.001. (C) Cell viability CCK-8 assay for cells treated with aspirin (200 µM), activated T cells (1:2 ratio to cancer cells), or the combination for 48 hours. Data are shown as the mean±SEM. *p<0.01. (D) T cell-mediated cancer cell-killing assay. PC-9OR and HCC827OR cells co-cultured in the indicated groups for 48 hours were subjected to crystal violet staining. Ratio of cancer cells to T cells: 2:1. (E) Ki67 incorporation assay on PC-9OR and HCC827OR cells treated as indicated. Activated T cells (1:2 ratio to cancer cells) or aspirin (200 µM) were added to the culture medium for 48 hours. Cells were then counterstained with DAPI. (F) Flow cytometry analysis of the exhaustion- and activation-related molecule Foxp3 in activated T cells co-cultured with the indicated cells (ratio of cancer cells to T cells: 1:1) for 48 hours with or without aspirin (200 µM). (G) PD-L1 levels in total protein extracts from indicated cells treated with aspirin for 48 hours, analyzed by western blotting. (H) LAMC2 levels in total protein extracts from the indicated cells treated with aspirin for 48 hours, analyzed by western blotting. (I) Immunofluorescence staining with an antibody against LAMC2 in PC-9 xenografts treated with osimertinib or osimertinib plus aspirin. (J) Schematic diagram of a T-cell chemotaxis assay directly regulated by the treatment of PC-9OR cells with aspirin (200 µM). Representative images of infiltrating T cells stained with CFSE dye. (K) Representative images of the immunofluorescence staining of CD68 (red), CD206 (green), and DAPI (blue) in mouse tumor tissue sections. (L) Immunofluorescence staining with antibodies against CD8 (red) and LAMC2 (green) in non-small cell lung cancer tissues treated with osimertinib or osimertinib plus aspirin. Scale bar: 50 µm.hu-PBMC,human-Peripheral blood mononuclear cell; S.C,Subcutaneous; CFSE, carboxyfluorescein diacetate succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; Foxp3, forkhead box P3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LAMC2, laminin subunit γ−2; PBS, phosphate-buffered saline; PD-L1, programmed death ligand 1.

Article Snippet: Then, the cells were fixed and incubated overnight with Ki67 (#M00254- 8, Boster, Wuhan, China).

Techniques: Injection, CCK-8 Assay, Cell Culture, Staining, Flow Cytometry, Activation Assay, Western Blot, Immunofluorescence, Chemotaxis Assay, Saline