control crispra plasmids  (Santa Cruz Biotechnology)

Journal: Science translational medicine

Article Title: Tumor-intrinsic NLRP3-HSP70-TLR4 axis drives premetastatic niche development and hyperprogression during anti–PD-1 immunotherapy

doi: 10.1126/scitranslmed.abq7019

Figure Lengend Snippet: (A) Representative example of frequencies of live+CD45+CD11b+Ly6G+ Ly6CloF4/80− PMN-MDSCs in the lungs of tumor-bearing and non-tumor-bearing autochthonous BRAFV600EPTEN−/− mice. PerCP, peridinin-chlorophyll-protein; FITC, fluorescein isothiocyanate. (B) qrt-PCR analysis of Cxcl5 and Cxcl2 expression by CD45+EpCAM− and CD45−EpCAM+ cell populations derived from the lung tissues of non–tumor-bearing and tumor-bearing BRAFV600EPTEN−/− mice (n = 3). Statistical analysis was performed by two-way ANOVA followed by Sidak’s multiple comparisons test. (C) Experimental schematic to investigate the role of tumor NLRP3 on lung PMN-MDSC accumulation. KD, knockdown; NTC, nontarget control. (D) Flow cytometry analysis of PMN-MDSCs in the lung tissues of non–tumor-bearing, BRAFV600EPTEN−/− tumor–bearing, and BRAFV600EPTEN−/−−NLRP3KD tumor–bearing mice (n = 3). Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparisons test. (E) qrt-PCR analysis of Cxcl1, Cxcl2, Cxcl3, and Cxcl5 expression in FACS-purified CD45−EpCAM+ lung epithelial cells derived from BRAFV600EPTEN−/− tumor–bearing and BRAFV600EPTEN−/−−NLRP3KD tumor–bearing mice (n = 3). (F) Experimental schematic to verify the role of tumor-intrinsic NLRP3 in metastatic progression. NLRP3i, NLRP3 inhibitor. (G) Flow cytometry analysis of PMN-MDSCs in lung tissues of BRAFV600EPTEN−/− tumor–bearing mice after treatment with either NLRP3i or vehicle control (Ctrl; n = 4). (H) Left: Low-magnification imaging and quantification of resected lung tissues after treatment with either NLRP3i or Ctrl. Images are shown at 4×. Scale bars, 2000 μm. Right: Survival curve analysis of BRAFV600EPTEN−/− tumor–bearing mice after treatment with either NLRP3i or Ctrl (n = 5). Statistical analysis for the right panel of (H) was performed by log-rank test. All two-group comparisons were analyzed using unpaired t tests. All data are representative of two to three independent experiments and expressed as mean values ± SEM. *P < 0.05, **P < 0.005, and ***P < 0.0005.

Article Snippet: The NLRP3 and control CRISPRa plasmids (Santa Cruz Biotechnology, sc-432122-ACT) were packaged into a lentiviral vector in human embryonic kidney 293T cells as previously prescribed ( 55 ).

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Knockdown, Control, Flow Cytometry, Purification, Imaging

Journal: Science translational medicine

Article Title: Tumor-intrinsic NLRP3-HSP70-TLR4 axis drives premetastatic niche development and hyperprogression during anti–PD-1 immunotherapy

doi: 10.1126/scitranslmed.abq7019

Figure Lengend Snippet: (A) Incidence of NLRP3 amplification in human tumor types based on The Cancer Genome Atlas (TCGA). Data were visualized using cBioPortal. (B) Flow cytometry analysis of PMN-MDSCs in the lungs of Ctrl and NLRP3a tumor–bearing mice (n = 3). (C) Left: S100β IHC of lungs derived from Ctrl and NLRP3a tumor–bearing mice. 20×; scale bars, 50 μm. Red arrows, S100β-positive cells. Right: Quantification of S100β+ cells in the lungs of Ctrl and NLRP3a tumor–bearing mice (n = 3). (D) Left: Flow cytometry analysis of lung tissues resected from TLR4+/+ control and SPC-TLR4−/− mice harboring control BRAFV600EPTEN−/− tumors or BRAFV600EPTEN−/−-NLRP3a tumors (n = 3). Right: Wnt5a qrt-PCR analysis of lung tissues from TLR4+/+ control and SPC-TLR4−/− mice harboring control BRAFV600EPTEN−/− tumors or BRAFV600EPTEN−/−-NLRP3a tumors (n = 3). (E) ELISA analysis of plasma HSP70 concentrations in Ctrl and NLRP3a tumor–bearing mice after IgG Ctrl or anti–PD-1 treatment (n = 3). Statistical analysis was performed by two-way ANOVA followed by Tukey’s multiple comparisons test. (F) Flow cytometry analysis of PMN-MDSCs in the lungs of Ctrl and NLRP3a tumor–bearing mice after anti–PD-1 treatment (n = 3). (G) Ctrl and NLRP3a TGRs after IgG Ctrl or anti–PD-1 treatment. Tumor growth is expressed as a ratio of anti–PD-1 treatment to IgG Ctrl treatment. All two-group comparisons were analyzed using unpaired t tests. All data are representative of two to three independent experiments and expressed as mean values ± SEM. *P < 0.05 and ***P < 0.0005.

Article Snippet: The NLRP3 and control CRISPRa plasmids (Santa Cruz Biotechnology, sc-432122-ACT) were packaged into a lentiviral vector in human embryonic kidney 293T cells as previously prescribed ( 55 ).

Techniques: Amplification, Flow Cytometry, Derivative Assay, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Science translational medicine

Article Title: Tumor-intrinsic NLRP3-HSP70-TLR4 axis drives premetastatic niche development and hyperprogression during anti–PD-1 immunotherapy

doi: 10.1126/scitranslmed.abq7019

Figure Lengend Snippet: (A) Baseline plasma HSP70 ELISA measurements in patients with advanced melanoma before initiating anti–PD-1 immunotherapy (n = 38). CR, complete responder; PR, partial responder; SD, stable disease; PD, progressive disease; HPD, hyperprogression disease. (B) NLRP3-ASC proximity ligation assay (PLA) analysis of baseline tumor tissues in patients with advanced melanoma before initiating anti–PD-1 immunotherapy (n = 34). Red dots represent NLRP3-ASC interactions. 40×; scale bars, 5 μm. For (A) and (B), statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparisons test. (C) Progression-free survival (PFS) analysis of patients with advanced melanoma stratified according to baseline tumor NLRP3-ASC PLA scores. Low NLRP3, below the NLRP3-ASC PLA median. High NLRP3, equal to or above the NLRP3-ASC PLA median. Statistical analysis was performed by log-rank test. All data are representative of two independent experiments and expressed as mean values ± SEM. *P < 0.05 and ***P < 0.0005.

Article Snippet: The NLRP3 and control CRISPRa plasmids (Santa Cruz Biotechnology, sc-432122-ACT) were packaged into a lentiviral vector in human embryonic kidney 293T cells as previously prescribed ( 55 ).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Proximity Ligation Assay