Journal: Cell

Article Title: Exosome RNA Unshielding Couples Stromal Activation to Pattern Recognition Receptor Signaling in Cancer

doi: 10.1016/j.cell.2017.06.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: This paper Table S8 Recombinant DNA Plasmid: pGFH-9 Katharina Strub Addgene #39538 Plasmid: pGFH-14c Katharina Strub Addgene #39541 Plasmid: pUltra-hot Malcom Moore Addgene #48139 Plasmid: pUltra-hot-SRP9-14 This paper N/A Plasmid: pOZ-N-FH Roger Greenberg N/A Plasmid: pOZ-N-RIG-I-WT This paper N/A Plasmid: pOZ-N-RIG-I-MUT This paper N/A Plasmid: pCMV-dR8.2 dvpr Bob Weinberg Addgene #8455 Plasmid: pCMV-VSV-G Bob Weinberg Addgene # 8454 Plasmid: PX458 Feng Zhang Addgene #48138 Plasmid: pCT-CD81-RFP SBI Cat#CYTO125-PA-1 Plasmid: pET His6 GST TEV LIC cloning vector (1G) Scott Gradia Addgene #29655 Plasmid: pET His6 GST TEV-SRP9 This paper N/A Software and Algorithms R language and environment for statistical computing and graphics https://www.r-project.org Bioconductor and packages https://www.bioconductor.org CRAN https://cran.r-project.org/web/packages/ cutadapt https://pypi.python.org/pypi/cutadapt bowtie2 http://bowtie-bio.sourceforge.net/bowtie2/index.shtml STAR https://github.com/alexdobin/STAR/releases Subread http://subread.sourceforge.net DESeq2 https://www.rdocumentation.org/packages/DESeq2 flowCore http://bioconductor.org/packages/release/bioc/html/flowCore.html Rtsne https://cran.r-project.org/web/packages/Rtsne/index.html mclust https://cran.r-project.org/web/packages/mclust/vignettes/mclust.html RNAstructure http://rna.urmc.rochester.edu/RNAstructure.html Open in a separate window KEY RESOURCES TABLE

Techniques: Virus, Recombinant, Imaging, Magnetic Beads, Staining, Gene Expression, RNA Sequencing, CRISPR, In Vitro, Plasmid Preparation, Cloning, Software

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: Imaging CAR-NK cells targeted to HER2 ovarian cancer with human sodium-iodide symporter-based positron emission tomography

doi: 10.1007/s00259-024-06722-w

Figure Lengend Snippet: ( A ) Lentiviral construct used to engineer SKOV3-ip1 ovarian cancer cells containing the human elongation factor 1 alpha promoter (pEF1a) to drive expression of tdTomato (tdT) and Firefly luciferase (Fluc) separated by a T2A peptide sequence. ( B ) Flow plots tdTomato expression in naïve SKOV3-ip1 and Fluc + tdT + SKOV3-ip1. ( C ) Flow plots showing Fluc + tdT + SKOV3-ip1 cells with or without HER2 immunostaining. ( D ) Lentiviral constructs used to engineer NK-92 cells. The first plasmid construct containing zsG and NIS gene separated by a T2A upstream of a pEF1a promoter was used for NK-92 transduction. The second plasmid contained a pEF1a promoter upstream of a HER2 CAR gene (containing DARPIN, CD8a, CD28, CD3ζ components), in addition to Antares, was used for the sequential transduction of NK-92. ( E-H ) Flow plots confirming the expression of zsG and/or Antares in NK-92 cells engineered with one or both constructs in D. I ) BLI images ( I ) and analysis ( J ) of cytotoxicity assays upon co-culturing of Fluc + tdT + SKOV3-ip1 with different NK cell phenotypes at various effector to target ratios

Article Snippet: Finally, a previously made pEF1α-tdT-Fluc lentiviral transfer plasmid containing a tdTomato (tdT) [pUltra-Chili-Luc was a gift from Malcolm Moore (Addgene plasmid # 48,688; http://n2t.net/addgene:48688 ; RRID: Addgene_48688)] and the BLI reporter gene Firefly luciferase (Fluc) under the control of pEF1 was used (previously cloned and described in [ ]).

Techniques: Construct, Expressing, Luciferase, Sequencing, Immunostaining, Plasmid Preparation, Transduction

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: Imaging CAR-NK cells targeted to HER2 ovarian cancer with human sodium-iodide symporter-based positron emission tomography

doi: 10.1007/s00259-024-06722-w

Figure Lengend Snippet: Tissue sections of tumor masses ( A-B ) and ovaries ( C-D ) stained with Hematoxylin and Eosin ( H & E ) of a control mouse and a mouse receiving the NIS + Antares + CAR NK therapy. Fluorescence images showing DAPI (nuclei; E-H ), TdTomato (Fluc + tdT + SKOV3-ip1 cells; I-L ), zsGreen (NIS + Antares + CAR NK; M-P ), and the merged images ( Q-T ). ZsGreen positive cells are shown on the periphery of the ovaries in mice receiving the NK therapy ( P and T ). Few zsG positive cells are seen infiltrating inside the ovaries

Article Snippet: Finally, a previously made pEF1α-tdT-Fluc lentiviral transfer plasmid containing a tdTomato (tdT) [pUltra-Chili-Luc was a gift from Malcolm Moore (Addgene plasmid # 48,688; http://n2t.net/addgene:48688 ; RRID: Addgene_48688)] and the BLI reporter gene Firefly luciferase (Fluc) under the control of pEF1 was used (previously cloned and described in [ ]).

Techniques: Staining, Control, Fluorescence

Journal: Cancers

Article Title: Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing

doi: 10.3390/cancers13205062

Figure Lengend Snippet: Intercellular calcium (Ca 2+ ) waves triggered by focal photodynamic therapy in vivo. ( A ) Shown are GCaMP6s fluorescence emission ( F ) variations (Δ F = F − F 0 , where F 0 = pre-stimulus value) at different time points after the onset of laser irradiation in standard conditions (normal extracellular medium containing 2 mM of Ca 2+ , NEM) or after 20 min incubation in Ca 2+ -free extracellular medium supplemented with ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, 5 mM). The contour of the irradiated cell is highlighted in the images captured at 5 s. Bystander cells were identified by ordinal numbers according to the distance from the irradiated cell (see, for an example, the contoured cells in the image at 70 s in EGTA conditions); scale bar: 20 µm; ( B ) Single-cell Δ F / F 0 traces [mean (solid lines) ± standard error of the mean (s.e.m., dashed lines)] generated as pixel signal average within regions of interest contouring the cell focal plane section for each bystander cell order (from 1st to 6th); pooled data from n ≥ 6 experiments in 3 tumors for both conditions: green traces, EGTA; blue traces, NEM; vertical dashed lines mark the onset of irradiation at t = 10 s; ( C ) Area under Δ F / F 0 curves ( A , inset) computed between t = 10 s and t = 80 s (mean ± s.e.m.) vs. bystander cell order (abscissa): green bars, EGTA; blue bars, NEM; a.u., arbitrary units; n.s., not significant; *, p < 0.05; **, p < 0.01; ***; p < 0.001; the Mann-Whitney U test.

Article Snippet: Lentiviral plasmid encoding GCaMP6s and mCherry were purchased from Addgene (Watertown, MA, USA; #80146, a gift from Dr. Darrell Kotton and pUltra-hot, and #24130, a gift from Dr. Malcolm Moore, respectively).

Techniques: In Vivo, Fluorescence, Irradiation, Incubation, Generated, MANN-WHITNEY

Journal: Cancers

Article Title: Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing

doi: 10.3390/cancers13205062

Figure Lengend Snippet: Connexin (Cx) hemichannels (HCs) expressed in melanoma cells mediate the propagation of calcium (Ca 2+ ) waves induced by focal photodynamic therapy (fPDT) in vivo. ( A ) Pooled results of fPDT trials in GCaMP6s-expressing dorsal skinfold chamber (DSC) tumors in the following conditions: Ca 2+ -free extracellular medium (CFEM) supplemented with ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, 5 mM); normal extracellular medium containing 2 mM of Ca 2+ (NEM, control); CFEM supplemented with EGTA (5 mM) plus carbenoxolone (CBX, 100 µM) or flufenamic acid (FFA, 100 µM) or TAT-Gap19 (150 µM) or abEC1.1m (1 μM). The histogram shows the area under GCaMP6s Δ F / F 0 traces ( A , inset) computed between the onset of fPDT ( t = 10 s) and the end of the observation time window ( t = 80 s) for each bystander cell order (abscissa); pooled data [mean ± standard error of the mean (s.e.m.)] for n ≥ 6 experiments in at least 2 different tumors for each condition. a.u., arbitrary units; *, p < 0.05; ***, p < 0.001, the Kruskal-Wallis test (for post hoc pairwise comparisons, see ). Data for EGTA and NEM conditions are also shown in C; ( B – D ) In vivo 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) uptake experiments: GCaMP6s-expressing DSC melanomas were incubated with DAPI (5 μM) dissolved in: CFEM supplemented with 5 mM of EGTA; NEM; CFEM supplemented with 5 mM of EGTA plus FFA (100 µM) or TAT-Gap19 (150 µM) or abEC1.1m (1 μM). Fluorescence images were acquired at 5 min intervals up to 30 min; ( B ) Representative images acquired before ( t = 0 min) and after 30 min of DAPI incubation in EGTA conditions; scale bar: 20 µm; ( C ) Relative variation of nuclear DAPI fluorescence intensity [ F ( t )/ F 0 ] in tumor cells vs. time during dye uptake in EGTA conditions (mean ± s.e.m., n = 20 cells, 2 tumors). The red dashed line was computed by data fitting with the shown function f ( t ) (parameter values: a = −0.1754, b = 0.0647 min −1 ); ( D ) Box plots showing the distributions of Δ F = F (30 min) − F 0 for DAPI measured in n ≥ 12 nuclei for each condition. Red horizontal bars indicate the median. ***, p < 0.001, the Kruskal-Wallis test (for post hoc pairwise comparisons, see ); ( E ) Representative western blots for Cx43 (top) and Cx26 (bottom) expression in tumors (T) derived from B16-F10 or B16-F10-GCaMP6s cells and grown in DSCs (denoted as B16-F10 T and B16-F10-GCaMP6s T, respectively) compared with B16-F10 or B16-F10-GCaMP6s cells grown in culture dishes; graphs on the right show the corresponding relative optical density (mean ± s.e.m., n = 4 independent experiments; **, p < 0.05, ANOVA on Ranks; ***, p < 0.001, ANOVA). Detailed information about the Western blotting can be found at . ( F ) Confocal fluorescence images obtained by immunostaining with antibodies selective for Cx43 (top left, green), Cx26 (bottom left, green) and MelanA (right, red) in representative sections of melanomas grown in DSCs; nuclei were stained with DAPI; scale bar: 10 μm.

Article Snippet: Lentiviral plasmid encoding GCaMP6s and mCherry were purchased from Addgene (Watertown, MA, USA; #80146, a gift from Dr. Darrell Kotton and pUltra-hot, and #24130, a gift from Dr. Malcolm Moore, respectively).

Techniques: In Vivo, Expressing, Control, Incubation, Fluorescence, Western Blot, Derivative Assay, Immunostaining, Staining

Journal: Cancers

Article Title: Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing

doi: 10.3390/cancers13205062

Figure Lengend Snippet: Whole-cell biosensors for adenosine triphosphate (ATP) detection (ATP-WCBs) are activated by extracellular ATP released during the propagation of calcium (Ca 2+ ) waves induced by focal photodynamic therapy (fPDT) in the dorsal skinfold chamber (DSC). ( A ) Schematic representation of the multiphoton microscope objective lens oscillating between two focal planes for real-time detection of ATP release during fPDT stimulation (left, GCaMP6s-expressing tumor; right, Fluo-8H-loaded ATP-WCBs); ( B ) Representative back-projections of Δ F = F − F 0 (with F 0 pre-stimulus value) frames acquired from tumor (top) and ATP-WCBs (bottom) during fPDT stimulation in the absence of apyrase (−APY, left) or in its presence (+APY, 250 U/mL, right) in Ca 2+ -free extracellular medium (CFEM). Red circles mark the location of the photoactivation laser beam; scale bar: 20 µm; ( C ) Average Ca 2+ responses of bystander melanoma cells (top) and ATP-WCBs (bottom) to fPDT before (left) and after (right) addition of APY to the extracellular medium; Δ F / F 0 signals [mean (solid lines) ± standard error of the mean (s.e.m., dashed lines)] are shown for each bystander cell order. Results are representatives of n ≥ 3 experiments performed in 2 tumors. Vertical dashed lines mark the onset of laser irradiation ( t = 5 s); ( D ) Effect of APY on the amplitudes of fPDT-induced Ca 2+ waves in CFEM supplemented with 5 mM of EGTA; the histogram shows the area under GCaMP6s Δ F / F 0 signals ( A , see inset) computed for each bystander cell order in the absence of APY (−APY, green bars, also shown in C and A) or in its presence (+APY, black bars); data (mean ± s.e.m.) were pooled from n ≥ 6 experiments in at least 2 different melanomas for each condition; a.u., arbitrary units; **, p < 0.01; ***, p < 0.001; the Mann-Whitney U test.

Article Snippet: Lentiviral plasmid encoding GCaMP6s and mCherry were purchased from Addgene (Watertown, MA, USA; #80146, a gift from Dr. Darrell Kotton and pUltra-hot, and #24130, a gift from Dr. Malcolm Moore, respectively).

Techniques: Microscopy, Expressing, Irradiation, MANN-WHITNEY