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nlrp3 and control crispra plasmids sc-432122-act  (Santa Cruz Biotechnology)


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    Santa Cruz Biotechnology nlrp3 and control crispra plasmids sc-432122-act
    (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 <t>NLRP3</t> 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.
    Nlrp3 And Control Crispra Plasmids Sc 432122 Act, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/nlrp3+and+control+crispra+plasmids/anti+nlrp3/pmc10347419-594-1-6
    Average 90 stars, based on 1 article reviews
    nlrp3 and control crispra plasmids sc-432122-act - by Bioz Stars, 2026-11
    90/100 stars

    Images

    1) Product Images from "Tumor-intrinsic NLRP3-HSP70-TLR4 axis drives premetastatic niche development and hyperprogression during anti–PD-1 immunotherapy"

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

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.abq7019

    (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.
    Figure Legend 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.

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

    (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.
    Figure Legend 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.

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

    (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.
    Figure Legend 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.

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

    Related Articles

    Control:

    Article Title: Tumor-intrinsic NLRP3-HSP70-TLR4 axis drives premetastatic niche development and hyperprogression during anti-PD-1 immunotherapy.
    Article Snippet: The tumor-intrinsic NOD-, LRRand pyrin domain-containing protein-3 (NLRP3) inflammasome–heat shock protein 70 (HSP70) signaling axis is triggered by CD8+ T cell cytotoxicity and contributes to the development of adaptive resistance to anti–programmed cell death protein 1 (PD-1) immunotherapy by recruiting granulocytic polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) into the tumor microenvironment.. Here, we demonstrate that the tumor NLRP3-HSP70 axis also drives the accumulation of PMN-MDSCs into distant lung tissues in a manner that depends on lung epithelial cell Toll-like receptor 4 (TLR4) signaling, establishing a premetastatic niche that supports disease hyperprogression in response to anti–PD-1 immunotherapy.. Lung epithelial HSP70-TLR4 signaling induces the downstream Wnt5a-dependent release of granulocyte colony-stimulating factor (G-CSF) and C-X-C motif chemokine ligand 5 (CXCL5), thus promoting myeloid granulopoiesis and recruitment of PMN-MDSCs into pulmonary tissues.

    Plasmid Preparation:

    Article Title: Tumor-intrinsic NLRP3-HSP70-TLR4 axis drives premetastatic niche development and hyperprogression during anti-PD-1 immunotherapy.
    Article Snippet: The tumor-intrinsic NOD-, LRRand pyrin domain-containing protein-3 (NLRP3) inflammasome–heat shock protein 70 (HSP70) signaling axis is triggered by CD8+ T cell cytotoxicity and contributes to the development of adaptive resistance to anti–programmed cell death protein 1 (PD-1) immunotherapy by recruiting granulocytic polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) into the tumor microenvironment.. Here, we demonstrate that the tumor NLRP3-HSP70 axis also drives the accumulation of PMN-MDSCs into distant lung tissues in a manner that depends on lung epithelial cell Toll-like receptor 4 (TLR4) signaling, establishing a premetastatic niche that supports disease hyperprogression in response to anti–PD-1 immunotherapy.. Lung epithelial HSP70-TLR4 signaling induces the downstream Wnt5a-dependent release of granulocyte colony-stimulating factor (G-CSF) and C-X-C motif chemokine ligand 5 (CXCL5), thus promoting myeloid granulopoiesis and recruitment of PMN-MDSCs into pulmonary tissues.



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    nlrp3 and control crispra plasmids sc-432122-act  (Santa Cruz Biotechnology)
    90
    Santa Cruz Biotechnology nlrp3 and control crispra plasmids sc-432122-act
    (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 <t>NLRP3</t> 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.
    Nlrp3 And Control Crispra Plasmids Sc 432122 Act, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/nlrp3+and+control+crispra+plasmids/anti+nlrp3/pmc10347419-594-1-6
    Average 90 stars, based on 1 article reviews
    nlrp3 and control crispra plasmids sc-432122-act - by Bioz Stars, 2026-11
    90/100 stars
    		  Buy from Supplier
    nlrp3 and control crispra plasmids  (Santa Cruz Biotechnology)
    90
    Santa Cruz Biotechnology nlrp3 and control crispra plasmids
    (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 <t>NLRP3</t> 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.
    Nlrp3 And Control Crispra Plasmids, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/nlrp3+and+control+crispra+plasmids/anti+nlrp3/pm36417489-361-1-6
    Average 90 stars, based on 1 article reviews
    nlrp3 and control crispra plasmids - by Bioz Stars, 2026-11
    90/100 stars
    		  Buy from Supplier

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    (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.

    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

    (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.

    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

    (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.

    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