Journal: Angiogenesis

Article Title: Imaging tumor angiogenesis in breast cancer experimental lung metastasis with positron emission tomography, near-infrared fluorescence, and bioluminescence

doi: 10.1007/s10456-013-9344-y

Figure Lengend Snippet: In vitro characterization of NOTA-TRC105-800CW and monitoring of fLuc-4T1 lung tumor burden with bioluminescence imaging. a Flow cytometry analysis of TRC105 and NOTA-TRC105-800CW in HUVECs (CD105 positive) and MCF-7 human breast cancer cells (CD105 negative) at multiple concentrations. Data from various control experiments are also shown. b Representative serial bioluminescence images of fLuc-4T1 lung tumor-bearing mice after intravenous injection of fLuc-4T1 cells. c Total photon flux of the bioluminescence signal (representing the total tumor burden) from the mouse lung (n = 8).

Article Snippet: Cell lines and animal model 4T1 murine BC, MCF-7 human BC, and human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: In Vitro, Imaging, Flow Cytometry, Control, Injection

Journal: Angiogenesis

Article Title: Imaging tumor angiogenesis in breast cancer experimental lung metastasis with positron emission tomography, near-infrared fluorescence, and bioluminescence

doi: 10.1007/s10456-013-9344-y

Figure Lengend Snippet: Serial in vivo PET/CT imaging of fLuc-4T1 lung tumor-bearing mice. a Serial coronal PET images at 4, 24, and 48 h post-injection of 64Cu-NOTA-TRC105-800CW, 64Cu-NOTA-cetuximab-800CW, or 64Cu-NOTA-TRC105-800CW after a 2 mg pre-injected dose of TRC105 (i.e., blocking). b Representative PET/CT images of fLuc-4T1 tumor-bearing mice in the three groups at 48 h post-injection. c Representative CT images of normal mouse lung, fLuc-4T1 lung metastasis, and a PET/CT fused image at 48 h post-injection of 64Cu-NOTA-TRC105-800CW into fLuc-4T1 lung tumor-bearing mice. Arrowheads indicate the fLuc-4T1 tumors.

Article Snippet: Cell lines and animal model 4T1 murine BC, MCF-7 human BC, and human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: In Vivo, Positron Emission Tomography-Computed Tomography, Imaging, Injection, Blocking Assay

Journal: Angiogenesis

Article Title: Imaging tumor angiogenesis in breast cancer experimental lung metastasis with positron emission tomography, near-infrared fluorescence, and bioluminescence

doi: 10.1007/s10456-013-9344-y

Figure Lengend Snippet: Quantitative analysis of the PET data. a Time-activity curves of the liver, fLuc-4T1 lung tumor, blood, and muscle upon intravenous injection of 64Cu-NOTA-TRC105-800CW. b Time-activity curves of the liver, fLuc-4T1 lung tumor, blood, and muscle upon intravenous injection of 64Cu-NOTA-TRC105-800CW, after a blocking dose of 2 mg of TRC105. c Time-activity curves of the liver, fLuc-4T1 lung tumor, blood, and muscle upon intravenous injection of 64Cu-NOTA-cetuximab-800CW. d Comparison of the fLuc-4T1 lung tumor uptake in the three groups. n = 3; **: P < 0.01.

Article Snippet: Cell lines and animal model 4T1 murine BC, MCF-7 human BC, and human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Activity Assay, Injection, Blocking Assay, Comparison

Journal: Angiogenesis

Article Title: Imaging tumor angiogenesis in breast cancer experimental lung metastasis with positron emission tomography, near-infrared fluorescence, and bioluminescence

doi: 10.1007/s10456-013-9344-y

Figure Lengend Snippet: Ex vivo imaging at 48 h post-injection of each tracer and correlation analysis. a Ex vivo bioluminescence imaging (BLI), near-infrared fluorescence (NIRF), and positron emission tomography (PET) imaging of major organs at 48 h post-injection of each tracer. Images are representative of 3 mice per group. T: fLuc-4T1 tumor-bearing lung, L: liver, B1: blood, H: heart, K: kidney, B2: bone, S: spleen, M: muscle. b Representative autoradiography and corresponding NIRF images of the entire fLuc-4T1 tumor-bearing lung tissue at 48 h post-injection of 64Cu-NOTA-TRC105-800CW. Arrowheads indicate tumor nodules. c Linear correlation of the total ex vivo NIRF signal, in all nine fLuc-4T1 tumor-bearing lung at 48 h post-injection, with the %ID/g values obtained from biodistribution studies (left) or analysis of the PET data (right).

Article Snippet: Cell lines and animal model 4T1 murine BC, MCF-7 human BC, and human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Ex Vivo, Imaging, Injection, Fluorescence, Positron Emission Tomography, Autoradiography

Journal: Angiogenesis

Article Title: Imaging tumor angiogenesis in breast cancer experimental lung metastasis with positron emission tomography, near-infrared fluorescence, and bioluminescence

doi: 10.1007/s10456-013-9344-y

Figure Lengend Snippet: a Biodistribution in fLuc-4T1 lung tumor-bearing mice at 48 h post-injection of 64Cu-NOTA-TRC105-800CW or 64Cu-NOTA-TRC105-800CW after a blocking dose of TRC105. b Biodistribution in fLuc-4T1 lung tumor-bearing mice at 48 h post-injection of 64Cu-NOTA-TRC105-800CW or 64Cu-NOTA-cetuximab-800CW. **: P < 0.01. n = 3.

Article Snippet: Cell lines and animal model 4T1 murine BC, MCF-7 human BC, and human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Injection, Blocking Assay

Journal: Angiogenesis

Article Title: Imaging tumor angiogenesis in breast cancer experimental lung metastasis with positron emission tomography, near-infrared fluorescence, and bioluminescence

doi: 10.1007/s10456-013-9344-y

Figure Lengend Snippet: Immunofluorescence staining of the fLuc-4T1 tumor lung, normal mouse lung, liver, and spleen tissue sections. Green: CD105; red: CD31; blue: DAPI. All images were acquired under the same conditions and displayed at the same scale. Magnification: 200×. Scale bar: 50 µm.

Article Snippet: Cell lines and animal model 4T1 murine BC, MCF-7 human BC, and human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Immunofluorescence, Staining

Journal: Angiogenesis

Article Title: Imaging tumor angiogenesis in breast cancer experimental lung metastasis with positron emission tomography, near-infrared fluorescence, and bioluminescence

doi: 10.1007/s10456-013-9344-y

Figure Lengend Snippet: Near-infrared fluorescence image-guided surgical removal of subcutaneous 4T1 tumors in nude mice, after intravenous injection of NOTA-TRC105-800CW. Mice were subjected to optical imaging at 4 and 24 h post-injection, immediately after the skin was open and tumor exposed, and after surgical removal of the tumor. Arrowheads indicate the 4T1 tumors in all cases.

Article Snippet: Cell lines and animal model 4T1 murine BC, MCF-7 human BC, and human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Fluorescence, Injection, Optical Imaging

Journal: bioRxiv

Article Title: Antigen-specific T cell immunotherapy by in vivo mRNA delivery

doi: 10.1101/2024.10.29.620946

Figure Lengend Snippet: (A) Schematic showing APN delivery of diverse mRNA cargo to cognate T cells. b-g, Activated NOD8.3 CD8 T cells were transfected in vitro with PBS, K d /Ctrl (non-cognate control) APNs, or K d /NRP-V7 (cognate) APNs. After 24 hours, transfection readout was measured. (B) T cells transfected by APNs carrying secreted nLuc mRNA were analyzed via IVIS and quantified. (C) Representative flow plots and frequency bar plot of intracellular BFP expression. (D) Representative flow plots and frequency bar plot of surface-bound VHH expression. One-way analysis of variance (ANOVA) and Tukey post-test and correction for multiple comparisons; ****P<0.0001. All data are means ± SD; n=3 independent wells. (E) Schematic showing the internalization mechanism of APN by T cells through T cell receptor (TCR), not low-density lipoprotein receptor (LDLR). (F) Activated NOD8.3 CD8 T cells were coincubated with either a LDLR blocking antibody (αLDLR Ab) or an isotype antibody control (isotype Ab). APNs were encapsulated with nLuc and transfection was measured by IVIS 24 hours post transfection. (G) Quantification of APN transfection in the presence of the TCR signaling inhibitor dasatinib (dasa), measured via flow cytometry. Two-way ANOVA with Sidak post-test and correction for multiple comparisons. NS= not significant; ****P<0.0001.

Article Snippet: Fluc activity was measured using an IVIS Spectrum CT (PerkinElmer) 10 min after intravenous injections after intraperitoneal injection of d-luciferin (15 mg/mL, 200 μL per mouse).

Techniques: Transfection, In Vitro, Control, Expressing, Blocking Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Antigen-specific T cell immunotherapy by in vivo mRNA delivery

doi: 10.1101/2024.10.29.620946

Figure Lengend Snippet: (A) - (C) Enriched human influenza A virus (IAV)-specific T cells from healthy donor were treated with APNs functionalized with human leukocyte antigen A2.1+ (HLA-A2.1) bound to IAV peptide (HLA/IAV APN). APNs were encapsulated with either nLuc or αBCMA CAR mRNA and transfection was analyzed through IVIS readout of nLuc (A) or flow cytometry detection of αBCMA CAR (B) to show transfection was limited to on-target IAV-specific T cells (C) . (D) - (E) , HLA/IAV APNs transfect human-IAV specific T cells with functional αBCMA CAR and kill luciferized MM.1R cancer cells in in vitro cocultures. (D) and quantified via bioluminescence IVIS readout (E) . One-way ANOVA with Tukey’s post-test and correction for multiple comparisons; means ± SD, n=3 independent wells. NS= not significant, ****P<0.0001. (F) - (G) , Frozen peripheral blood mononuclear cells (PBMC) were obtained from HLA-A2.1 positive MM patients who were previously treated with daratumumab and peptide pulsed with peptide epitopes (F) , leading to IAV and CMV-specific T cell expansion (G) . (H) Human MM patient CMV- and IAV-specific T cells were mixed together and transfected in vitro with HLA/IAV and HLA/CMV APNs encapsulated with αBCMA CAR and αGPRC5D CAR mRNA respectively. Two-way ANOVA with Sidak post-test and correction for multiple comparisons; means ± SD, n=3 independent wells. NS = not significant, ***P<0.001, ****P<0.0001.

Article Snippet: Fluc activity was measured using an IVIS Spectrum CT (PerkinElmer) 10 min after intravenous injections after intraperitoneal injection of d-luciferin (15 mg/mL, 200 μL per mouse).

Techniques: Virus, Transfection, Flow Cytometry, Functional Assay, In Vitro

Journal: bioRxiv

Article Title: Antigen-specific T cell immunotherapy by in vivo mRNA delivery

doi: 10.1101/2024.10.29.620946

Figure Lengend Snippet: (A)-(C) Enriched human I nfluenza A virus (IAV)-specific T cells from a healthy donor were intravenously injected into NSG mice and dosed with HLA/IAV APNs carrying αBCMA CAR mRNA (A) and IAV-specific T cells in the spleen were quantified (B) . Student’s t-test; means ± SD, n=4-5 biological replicates. NS= not significant. (C) I n vivo transfection efficiency with HLA/IAV APNs and HLA/CMV APNs carrying αBCMA CAR mRNA was analyzed via flow cytometry. One-way ANOVA with Tukey’s post-test and correction for multiple comparisons; means ± SD, n= 7 biological replicates. ****P<0.0001. (D) NSG mice were intravenously inoculated with luciferized human BCMA+ U266 MM tumor cells, injected with enriched human IAV-specific T cells, and dosed with HLA/IAV APNs encapsulated with αBCMA CAR mRNA. Tumor burden was measured via luminescence by IVIS (E) and quantified up to 30 days after tumor inoculation (F) . Two-way ANOVA with Sidak post-test and correction for multiple comparisons; means ± SEM, n=5 biological replicates. NS = not significant, ***P<0.001, ****P<0.0001.

Article Snippet: Fluc activity was measured using an IVIS Spectrum CT (PerkinElmer) 10 min after intravenous injections after intraperitoneal injection of d-luciferin (15 mg/mL, 200 μL per mouse).

Techniques: Virus, Injection, Transfection, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Co-targeting RANK pathway treats and prevents acquired resistance to CDK4/6 inhibitors in luminal breast cancer

doi: 10.1016/j.xcrm.2023.101120

Figure Lengend Snippet:

Article Snippet: To quantify cell viability, cells were seeded in 96-well plates at a density of 1 × 10 4 cells/well, with or without fulvestrant (Selleck Chemicals), palbociclib (Sigma-Aldrich), ribociclib (Santa Cruz Biotechnology), abemaciclib (Selleck Chemicals), OPG-Fc (Amgen Inc), RANK-Fc (R&D Systems), 3-ATA (Santa Cruz Biotechnology), seliciclib (Focus Biomolecules), and/or fludarabine (Santa Cruz Biotechnology).

Techniques: Recombinant, Control, shRNA, Blocking Assay, Alamar Blue Assay, Flow Cytometry, Isolation, cDNA Synthesis, Bicinchoninic Acid Protein Assay, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Gene Expression, Western Blot, Software

Journal: Nature biomedical engineering

Article Title: Gold/alpha-lactalbumin nanoprobes for the imaging and treatment of breast cancer

doi: 10.1038/s41551-020-0584-z

Figure Lengend Snippet: a, Schematic of the NIR fluorescent AuQC705 nanoprobe in ultrasmall size synthesized from gold precursors and milk protein α-LA intravenously injected through the tail vein of a mouse bearing a human breast tumour. AuQC705 circulates into the cardiac blood pool (i) and passively targets the tumour (ii) through its highly permeable angiogenic vasculatures for imaging. The high specificity of passive tumour targeting and retention results from the combined effects of malformed leaky tumour vasculatures and poor extravasation of impaired lymphatic vessels, which are both absent in healthy tissues. Excessive AuQC705 is cleared from the bloodstream by glomerular filtration of renal tubules (iii) and urinary excretion of the bladder (iv). HD, hydrodynamic size. b, In the well-organized α-LA framework that dictates its biological identity and governs molecular pathways, AuQC705 permeates the tumour via fenestrations in arterioles and capillaries while barely penetrating normal blood vessels which lack such fenestrations. Endocytosis of AuQC705 is mediated by macropinocytosis of cancer cells, which sustains survival and proliferation using extracellular proteins as nutrient pathways in a series of steps, including cytoskeleton rearrangement and membrane ruffling (i); ruffle fold-back and engulfment (ii); irregularly shaped large macropinosome vesicle trafficking (iii); and lysosomal catabolism (iv). c, Gold with high Z and high K-edge absorption (orange line) provides superior CT contrast compared with clinically approved iodine (black line) in mammography and clinical CT windows. Data were retrieved from the National Institute of Standards and Technology. μen ρ–1, mass energy-absorption coefficient. d, Absorption spectra of major endogenous chromophores in breast tissues, assuming an artery–vein distribution of 70% tissue oxygen saturation94. The optical imaging window of AuQC705 is optimal, with an excitation (Ex) wavelength of 500 nm in the visible region and an emission (Em) wavelength of 700 nm in the NIR region in which HbO2–Hb, lipids and water have low absorption, together with a large Stokes shift that substantially reduces autofluorescence.

Article Snippet: Serial dilutions of the goat anti-bovine-α-LA antibodies (A10-128, Bethyl) were used as the positive control and 1 M NaCl buffer was used for regeneration.

Techniques: Synthesized, Injection, Imaging, Filtration, Optical Imaging

Journal: Nature biomedical engineering

Article Title: Gold/alpha-lactalbumin nanoprobes for the imaging and treatment of breast cancer

doi: 10.1038/s41551-020-0584-z

Figure Lengend Snippet: a, Excitation (dotted lines) and emission (solid lines; excitation, 360 nm) spectra of AuQCs demonstrating maximum excitation peaks at 370–380 nm and emission peaks at 450 nm, 520 nm and 705 nm. A secondary excitation at 520 nm for AuQC705 was identified (arrow). b, The size distribution of AuQCs, showing core sizes measured using TEM and hydrodynamic diameters measured using dynamic light scattering in PBS. Zeta potentials and polydispersity index (PDI) are indicated. c, The photostability of AuQCs at 360 nm continuous exposure. d, Single-excitation optical colour coding by AuQCs. The photographs were taken under 302 nm ultraviolet excitation (top) and white light (bottom). Multiplexed intensities can realize theoretically mixed RGB colours. 000, deionized water without AuQCs. NA, not applicable. e, In vitro single-excitation multiplexed fluorescence imaging. Images were multispectrally unmixed with spectra of AuQCs and coded in RGB pseudocolours. f, CIE 1931 xy chromaticity diagram showing the coordinates and correlated colour temperatures (CCT) of AuQCs with a large gamut. g, Far-ultraviolet CD spectra of AuQCs and native α-LA. Secondary-structure compositions were determined using BeStSel. Apo-state bovine α-LA acquired from RSCB (Protein Data Bank, 1F6R) was quantified and matched the experimental data well. Δε, molar circular dichroism. h, In vivo single-excitation multiplexing using AuQCs in a J:NU mouse after subcutaneous injection. The unit of radiant efficiency is photons s−1 cm−2 sr−1 (μW cm−2)−1. Specific in vivo spectra of AuQCs can be unmixed from tissue autofluorescence and are close to in vitro spectra (Supplementary Fig. 9b). The ratios of averaged specific fluorescence to background were calculated and compared. Excitation wavelength, 430 nm.

Article Snippet: Serial dilutions of the goat anti-bovine-α-LA antibodies (A10-128, Bethyl) were used as the positive control and 1 M NaCl buffer was used for regeneration.

Techniques: In Vitro, Fluorescence, Imaging, In Vivo, Multiplexing, Injection

Journal: Nature biomedical engineering

Article Title: Gold/alpha-lactalbumin nanoprobes for the imaging and treatment of breast cancer

doi: 10.1038/s41551-020-0584-z

Figure Lengend Snippet: a, Fluorescence spectra of AuQC705, AuQC705 after 10 min heat shock at 70 °C and AuQC705–BAMLET at pH 7 (left). Inset: decrease in optical transparency after formation of AuQC705–BAMLET. Right, ANS spectra of α-LA, AuQC705 and AuQC705–BAMLET, reflecting surface hydrophobicity. a.u., arbitrary units. b, The viability of MDA-MB-231 cancer cells after 3 h treatment of BAMLET (2 mg ml−1), α-LA (2 mg ml−1), AuQC705 (100 μg ml−1) and AuQC705–BAMLET (100 μg ml−1); n = 3 biologically independent samples. For the positive control, 1 mM SDS was used. c, Flow cytometry analysis of MDA-MB-231 cells that were stained with annexin V-CF488 (excitation, 488 nm; emission, 530/50 nm band pass (BP)) and EthD-III (excitation, 532 nm; emission, 610/20 nm BP) for early apoptosis and late apoptosis/necrosis, respectively, after 3 h treatment of α-LA, AuQC705 and AuQC705–BAMLET (75 μg ml−1). Staurosporine (5 μM) was used as an apoptosis-inducing control. The percentage of cells in each quadrant is indicated. d, Time-lapse DIC microscopy of MDA-MB-231 cells treated with 250 μg ml−1 AuQC705–BAMLET (top). Bottom, morphological and intracellular fluorescence analysis of a single cell. Scale bars, 20 μm (top row) and 10 μm (bottom three rows). e, Cell cycle analysis by quantifying DNA content using the intracellular DNA-binding dye propidium iodide (excitation, 532 nm; emission, 610/20 nm BP); n = 3 biologically independent samples. MDA-MB-231 cancer cells were treated with 2 mg ml−1 α-LA, 100 μg ml−1 AuQC705 and 5–100 μg ml−1 AuQC705–BAMLET for 1 h. The percentage of cells is indicated on the plot for cell cycle phases of sub-G1, G0/G1, S and G2/M corresponding to DNA content of <2 N, 2 N, 2N–4N and 4 N, respectively (left). Right, the percentage of each cell cycle phase. f, Flow cytometry analysis of mitochondrial membrane potentials (ΔΨM) using lipophilic cationic JC-1 with 100 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) as a positive control; n = 3 biologically independent samples. The top left gate represents normal mitochondria in healthy cells with high ΔΨM, intense in red fluorescence (excitation, 532 nm; emission, 610/20 nm BP) from JC-1 aggregates. The cell population in the bottom right gate has low ΔΨM with green fluorescence (excitation, 488 nm; emission, 530/50 nm BP) from monomeric JC-1. The schematic shows the shift of red fluorescence of JC-1 aggregates into green fluorescence of JC-1 monomers by AuQC705–BAMLET, causing mitochondrial depolarization (bottom left). The ratio of JC-1 aggregates to monomers (red/green fluorescence ratio) is a sensitive indicator of mitochondrial membrane polarization (bottom right). g, Cell energy phenotype diagram showing phenotypic switching of MDA-MB-231 cells from the baseline control by AuQC705 and AuQC705–BAMLET; n = 2 biologically independent samples. OCR, oxygen consumption rate; ECAR, extracellular acidification rate. h, Tumour growth patterns of MDA-MB-231 human xenografts co-implanted with AuQC705–BAMLET. AuQC705–BAMLET showed statistically significant growth inhibition compared with the control group, whereas no significance was found for either α-LA or AuQC705; n = 3 biologically independent animals. i, Localized anti-cancer therapy guided by NIR-fluorescence images in dual orthotopic MDA-MB-231/468 breast cancer models using AuQC705–BAMLET. The unit of radiant efficiency is photons s−1 cm−2 sr−1 (μW cm−2)−1. No AuQC705–specific signals were detected at the baseline. AuQC705–BAMLET induced potent cell death and was confined within the tumours, as visualized by fluorescence. Spatially defined drug distribution within the tumours was revealed by ex vivo microscopic imaging after identical in vivo imaging patterns were observed in both of the tumours, and was in agreement with the histopathology analysis. Newly differentiated viable cancer cells have low AuQC705 signals, whereas strong signals from AuQC705–BAMLET correlate well with cell death. One enlarged region of cell death is presented for each tumour. H&E, haematoxylin and eosin. Scale bars, 1,000 μm (left), 2,000 μm (middle) and 100 μm (right).

Article Snippet: Serial dilutions of the goat anti-bovine-α-LA antibodies (A10-128, Bethyl) were used as the positive control and 1 M NaCl buffer was used for regeneration.

Techniques: Fluorescence, Positive Control, Flow Cytometry, Staining, Microscopy, Cell Cycle Assay, Binding Assay, Inhibition, Ex Vivo, Imaging, In Vivo Imaging, Histopathology

Journal: Nature biomedical engineering

Article Title: Gold/alpha-lactalbumin nanoprobes for the imaging and treatment of breast cancer

doi: 10.1038/s41551-020-0584-z

Figure Lengend Snippet: a, The effects of pharmacological inhibitors; n = 3 biologically independent samples. b, Flow cytometry analysis corresponding to a with typical single-parameter gating for approximately 50% of the positive control without inhibitors shows inhibition of internalization. c, Direct and competitive uptake assays of AuQC705 in MDA-MB-231 cells demonstrated typical features of macropinocytosis; n = 3 biologically independent samples. While increasing concentrations of AuQC705 led to increased uptakes, no obvious blocking effects were observed in the presence of competitive α-LA. d, A three-dimensionally (3D) rendered image of a confocal z stack of MDA-MB-231 cells overlaid with a bright-field image. AuQC705 (red) and the macropinocytosis marker FITC–dextran (green) showed a high degree of cytosolic colocalization (yellow) at 2 h. Scale bar, 10 μm. A complete time course for organelle trafficking is shown in Supplementary Fig. 18. e, Ultrastructure analysis with TEM showing an early-formed multivesicular non-homogeneously large-sized (>0.2 μm) and irregularly shaped macropinosome vesicle in MDA-MB-231 cells after 1 h uptake. The cargo is close to the plasma membrane, indicating membrane closure and separation. AuQC705 maintained in ultrasmall size can be clearly observed as black dots in the vesicle without apparent aggregation, thereby preserving fluorescence. Scale bars, 2 μm (left), 200 nm (middle) and 50 nm (right). f, Immunofluorescence images of MDA-MB-231 tumours from a J:NU mouse 1 h after injection of AuQC705; DAPI was used to visualize nuclei (blue). The organelle trafficking of AuQC705 (red) and intracellular early endosome–macropinosome marker EEA1 (green) were in punctate patterns. Colocalizations are indicated by white arrows. Scale bars, 20 μm. g, The majority of AuQC705 (red) was found to end up in late endosomes and lysosomes in tumours 2 h after injection, as observed from the extensive colocalization (yellow) with LAMP1 (green). Scale bars, 100 μm.

Article Snippet: Serial dilutions of the goat anti-bovine-α-LA antibodies (A10-128, Bethyl) were used as the positive control and 1 M NaCl buffer was used for regeneration.

Techniques: Flow Cytometry, Positive Control, Inhibition, Blocking Assay, Marker, Preserving, Fluorescence, Immunofluorescence, Injection

Journal: PLoS ONE

Article Title: Fn14•Trail Effectively Inhibits Hepatocellular Carcinoma Growth

doi: 10.1371/journal.pone.0077050

Figure Lengend Snippet: (A) The amino-acid sequence of the Fn14-TRAIL protein. The amino-acid sequence of the extra-cellular domain of human Fn14 (amino-acids 1-52 of the mature protein, marked in bold letters) are directly linked to the extra-cellular domain of human TRAIL (amino-acids 53-217 of the mature protein, non-bold letters). The underlined sequence represents the signal-peptide of the human Urokinase protein, utilized to secrete Fn14-TRAIL out of the cell and removed from the mature protein. (B) Fn14-TRAIL separated at denaturizing conditions on SDS-PAGE, Coomassie gel staining. (C) Western blot analysis with anti-TRAIL and anti-Fn14 primary antibodies.

Article Snippet: Quantitive real-time PCR was performed using TaqMan Assay-on-Demand TM (TWEAK - Hs00356411_m1, Fn14 - Hs00171993_m1, TRAIL - Hs00234355_m1, DR4 - Hs00269492_m1, DR5 - Hs01043171_m1, DcR1 - Hs00182570_m1, DcR2 - Hs01043162_g1, OPG - Hs00171068_m1) and TaqMan Gene Expression Master Mix (Applied Biosystems).

Techniques: Sequencing, SDS Page, Staining, Western Blot

Journal: PLoS ONE

Article Title: Fn14•Trail Effectively Inhibits Hepatocellular Carcinoma Growth

doi: 10.1371/journal.pone.0077050

Figure Lengend Snippet: ( A ) The mRNA expression level of TRAIL, TRAIL receptors (DR4, DR5, DCR-1, DCR-2, OPG), Fn14 and TWEAK was determined by quantitive real-time PCR analysis. A representative experiment of three independent experiments is shown. Data are shown as average of triplicates (SD < 0.3), normalized against two endogenous control human genes, TBP and Actin-B, as calculated by Dataassist v2.0 software. ( B , C ) Protein expression of TRAIL, TRAIL receptors (DR4, DR5, DcR1, DcR2), Fn14 and TWEAK was determined by flow cytometeric analysis. ( D ) Fn14•TRAIL binds to HCC cells – HepG2 cells were incubated with Fn14•TRAIL, soluble TRAIL, Fn14 or the combination of the latter for 30 min at 4°c, immune-stained with PE-conjugated anti-Fn14, and analyzed by flow-cytometry. The results represent the mean +/- SD of triplicates (* p ≤ 0.05).

Article Snippet: Quantitive real-time PCR was performed using TaqMan Assay-on-Demand TM (TWEAK - Hs00356411_m1, Fn14 - Hs00171993_m1, TRAIL - Hs00234355_m1, DR4 - Hs00269492_m1, DR5 - Hs01043171_m1, DcR1 - Hs00182570_m1, DcR2 - Hs01043162_g1, OPG - Hs00171068_m1) and TaqMan Gene Expression Master Mix (Applied Biosystems).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Software, Incubation, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Fn14•Trail Effectively Inhibits Hepatocellular Carcinoma Growth

doi: 10.1371/journal.pone.0077050

Figure Lengend Snippet: ( A ) SK-HEP-1 [A, left panel], HepG2 [A, middle panel] and Huh7 [A, right panel] HCC cell lines, as well as NKNT3 [B, left panel] and FHB [B, right panel] hepatocyte cell lines, were incubated with 0, 3, 30 or 300 ng/ml of Fn14•TRAIL, TRAIL, Fn14-Fc or combination of the later two for 48 hours. Viable cells were stained with trypan blue and counted. The results represent the mean +/- SD of three independent experiments (* p ≤ 0.05). ( B ) HepG2 HCC cells were incubated with 30ng/ml Fn14•TRAIL for 24 hours, in the presence or absence of anti Fn14 or anti TRAIL blocking antibodies. Treated cells were stained by Annexin V-FITC and Propidium Iodide, and counted by flow cytometer (2x10 4 cells per sample). The results represent the mean +/- SD of two independent experiments (* p ≤ 0.05).

Article Snippet: Quantitive real-time PCR was performed using TaqMan Assay-on-Demand TM (TWEAK - Hs00356411_m1, Fn14 - Hs00171993_m1, TRAIL - Hs00234355_m1, DR4 - Hs00269492_m1, DR5 - Hs01043171_m1, DcR1 - Hs00182570_m1, DcR2 - Hs01043162_g1, OPG - Hs00171068_m1) and TaqMan Gene Expression Master Mix (Applied Biosystems).

Techniques: Incubation, Staining, Blocking Assay, Flow Cytometry

Journal:

Article Title: The Death Ligand TRAIL in Diabetic Nephropathy

doi: 10.1681/ASN.2007050581

Figure Lengend Snippet: TRAIL and OPG array expression correlate with severity of human DN. The panels show the correlation between TRAIL and OPG array expression and diverse parameters of severity of DN. Top and bottom left panels show the correlation between TRAIL and OPG gene expression in arrays and real-time qRT-PCR. TRAIL and OPG array mRNA data are expressed as Log2 signal intensity values. Linear regression was used to perform the best fit lines.

Article Snippet: 45 TaqMan reagents were used for TRAIL (Hs00234356_m1; {"type":"entrez-nucleotide","attrs":{"text":"NM_003810","term_id":"300193030","term_text":"NM_003810"}} NM_003810 ) and OPG (Hs 00171068_m1; MN_002546; Applied Biosystems, Darmstadt, Germany).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR

Journal:

Article Title: The Death Ligand TRAIL in Diabetic Nephropathy

doi: 10.1681/ASN.2007050581

Figure Lengend Snippet: Tubular epithelial TRAIL expression is regulated by proinflammatory cytokines. (A) Expression of TRAIL and OPG in HK-2 cells assessed by Western blot. (B) Semiquantitative RT-PCR. Expression plasmids for TRAIL-R1, -R2, and -R3 were used as controls. RT-PCR for TRAIL-R2 yielded two bands corresponding to its two transcript variants. (C and D) TRAIL protein expression in HK-2 cells. Cells were cultured in 5.5 or 25 mM glucose (C) or in 11 mM glucose and treated with a combination of cytokines (TNF-α and IFN-γ; D). Data are means ± SEM of three independent experiments. *P < 0.05. ADU, arbitrary densitometry units.

Article Snippet: 45 TaqMan reagents were used for TRAIL (Hs00234356_m1; {"type":"entrez-nucleotide","attrs":{"text":"NM_003810","term_id":"300193030","term_text":"NM_003810"}} NM_003810 ) and OPG (Hs 00171068_m1; MN_002546; Applied Biosystems, Darmstadt, Germany).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal:

Article Title: The Death Ligand TRAIL in Diabetic Nephropathy

doi: 10.1681/ASN.2007050581

Figure Lengend Snippet: High glucose sensitizes cells to TRAIL-induced cell death. (A) Apoptosis induction. Dose-response at 24 h. ▪, Cells cultured in 5.5 mM glucose; □, cells cultured in 25 mM glucose. *P < 0.05 versus absence of TRAIL 5.5 mM glucose; **P < 0.05 versus absence of TRAIL 25 mM glucose. Note the difference in the scale from B. (B) Apoptosis of HK2-cells treated with TRAIL 10 ng/ml and cytokines (TNF-α and IFN-γ) for 24 h. ▪, Cells cultured in 5.5 mM glucose; □, cells cultured in 25 mM glucose. Quantification of apoptosis by flow cytometry of DNA content. Experiments were performed five times, and each experiment consisted of triplicates. *P < 0.05. (C) Representative flow cytometry diagrams of cells cultured in 25 mM glucose. The line encompasses hypodiploid cells.

Article Snippet: 45 TaqMan reagents were used for TRAIL (Hs00234356_m1; {"type":"entrez-nucleotide","attrs":{"text":"NM_003810","term_id":"300193030","term_text":"NM_003810"}} NM_003810 ) and OPG (Hs 00171068_m1; MN_002546; Applied Biosystems, Darmstadt, Germany).

Techniques: Cell Culture, Flow Cytometry

Journal:

Article Title: The Death Ligand TRAIL in Diabetic Nephropathy

doi: 10.1681/ASN.2007050581

Figure Lengend Snippet: TRAIL induces activation of NF-κB in tubular epithelial cells. (A) NF-κB DNA binding is detected by electrophoretic mobility shift assay after TRAIL stimulation (10 ng/ml). (B) Western blot. Parthenolide (P) induces transient accumulation of IκBα and prevents TRAIL-induced IκBα degradation. The fragment corresponding to activated caspase-3 can be detected in cells treated with parthenolide and TRAIL. B, basal. (C) Parthenolide (P) enhances TRAIL-induced apoptosis. Apoptotic cells quantified by flow cytometry of cell DNA content. The pictures show apoptotic nuclei (white arrows) present in permeabilized, propidium iodide–stained cells treated with parthenolide and TRAIL for 24 h. Data are means ± SEM of five independent experiments. *P < 0.05 versus other groups. Magnification, ×40.

Article Snippet: 45 TaqMan reagents were used for TRAIL (Hs00234356_m1; {"type":"entrez-nucleotide","attrs":{"text":"NM_003810","term_id":"300193030","term_text":"NM_003810"}} NM_003810 ) and OPG (Hs 00171068_m1; MN_002546; Applied Biosystems, Darmstadt, Germany).

Techniques: Activation Assay, Binding Assay, Electrophoretic Mobility Shift Assay, Western Blot, Flow Cytometry, Staining

Journal:

Article Title: The Death Ligand TRAIL in Diabetic Nephropathy

doi: 10.1681/ASN.2007050581

Figure Lengend Snippet: OPG interferes with TRAIL-induced NF-κB activation and TRAIL-induced loss of tubular cell survival in vitro. Recombinant OPG (rOPG) acts as a soluble ligand for TRAIL, blocking its binding to the target cells, and anti-OPG blocking antibodies (αOPG) inhibit OPG blockade of TRAIL, thus enhancing its activity on target cells. (A) Quantitative analysis of NF-κB activation by luciferase assay. RLU, relative light units. *P < 0.05 versus basal unstimulated; #P < 0.01. Experiments were conducted three or more times. Samples were prepared at least in triplicate. (B) Cell survival was assessed using MTS. P, parthenolide. *P < 0.05 versus all other groups; **P < 0.05 versus basal control. Experiments were conducted six times. Samples were prepared at least in triplicate. Data are means ± SEM. (C through F). Proposed interaction between TRAIL and OPG. (C) TRAIL induces a low level of apoptosis in cultured tubular epithelial cells. The concurrent activation of NF-κB by TRAIL contributes to the low level of apoptosis, because the NF-κB inhibitor parthenolide sensitizes to TRAIL-induced apoptosis (D). (E) OPG is a soluble decoy receptor for TRAIL and is able to block both apoptosis and NF-κB activation in response to TRAIL. (F) OPG limits the increased apoptosis induced by TRAIL when NF-κB is inhibited. We propose that in vivo the relative local concentrations of TRAIL versus OPG in the cell microenvironment determines the outcome of the interaction.

Article Snippet: 45 TaqMan reagents were used for TRAIL (Hs00234356_m1; {"type":"entrez-nucleotide","attrs":{"text":"NM_003810","term_id":"300193030","term_text":"NM_003810"}} NM_003810 ) and OPG (Hs 00171068_m1; MN_002546; Applied Biosystems, Darmstadt, Germany).

Techniques: Activation Assay, In Vitro, Recombinant, Blocking Assay, Binding Assay, Activity Assay, Luciferase, Control, Cell Culture, In Vivo

Journal:

Article Title: The Death Ligand TRAIL in Diabetic Nephropathy

doi: 10.1681/ASN.2007050581

Figure Lengend Snippet: The diabetic milieu renders cells more sensitive to TRAIL-induced apoptosis. Diagram representing the hypothetical role of TRAIL in human DN. In the normal kidney, tubular cells express low levels of TRAIL that may regulate inflammation and tumor transformation. During diabetes, TRAIL expression is increased, probably as a consequence of a proinflammatory milieu, sensitizing cells to TRAIL-induced apoptosis.

Article Snippet: 45 TaqMan reagents were used for TRAIL (Hs00234356_m1; {"type":"entrez-nucleotide","attrs":{"text":"NM_003810","term_id":"300193030","term_text":"NM_003810"}} NM_003810 ) and OPG (Hs 00171068_m1; MN_002546; Applied Biosystems, Darmstadt, Germany).

Techniques: Transformation Assay, Expressing

Journal: bioRxiv

Article Title: EGFR + lung adenocarcinomas coopt alveolar macrophage metabolism and function to support EGFR signaling and growth

doi: 10.1101/2023.04.15.536974

Figure Lengend Snippet: Schematic of lung adenocarcinoma (LUAD) induction and immune profiling in a genetically inducible EGFR L858R mouse LUAD model. Tumors were initiated via feeding LUAD mice ( Ccsp-rtTA; TetOEGFR L858R ) or littermate control ‘Healthy’ mice ( TetO-EGFR L858R or Ccsp-rtTA ) doxycycline (DOX) in chow diet. Unless otherwise noted, mice were analyzed 6-8 weeks on DOX. (B) Serial MRI of a mouse after 4 and 6 or 7 weeks on DOX. (C) Immune infiltrates were quantified by flow cytometry during disease initiation (2-3 weeks), at emergence of macroscopic disease (4 weeks) and with fully established disease (6-8 weeks). Alveolar macrophages (AMs) were defined as CD45 + CD11b - SigF + CD11c + and interstitial macrophages (IMs) were defined as CD45 + AM - CD11b + Ly6G - . CD4 + and CD8 + T cells were gated on CD45 + CD3 + cells. (D) as in C, but AMs were stimulated with LPS and T cells were stimulated with PMA and ionomycin for 6 hrs prior to intracellular TNF staining. (E-F) After 6-8 weeks on DOX, AMs were isolated from LUAD mice (red) or littermate controls (black) and amounts of the inhibitory receptor CD200R (E) or checkpoint ligand PD-L1 (F) were measured by flow cytometry based on mean fluorescent intensity (MFI). (G) Bulk RNA-sequencing of AMs from late stage EGFR L858R lung tumors were analyzed for expression of several pro-inflammatory cytokines (n=3). Data from Ayeni et al. 2019. (H-I) LUAD mice were administered clodronate liposomes 2× weekly retroorbitally at weeks 4-6 of DOX and then sacrificed at week 6 of Dox. AM frequency was measured by flow cytometry (H) or tumor burden was assessed by the dry lung weight (I). (J) AM proliferation rates were assessed by Ki67-staining and flow cytometry. (K) GM-CSF was measured in healthy and LUAD lung lysates using ELISA. (L) AM surface expression of GM-CSFR was measured using flow cytometry. (M) Representative flow plots showing intracellular GM-CSF and Ki67 in healthy and malignant AT2 cells (CD45 - Epcam + CD31 - MHCII hi proSPC + ) incubated with BFA for 6 hours. (N-P) GM-CSF blocking (blue) or isotype control mAbs (red) were administered twice weekly (0.5mg/mouse) i.p. to LUAD mice during weeks 4-7 on DOX. We then measured the number of TA-AMs present in the BALF fluid (N), tumor burden using lung dry weight as a surrogate (O), and AM TNF secretion following LPS stimulation (P). Data shown are mean ± SEM, and statistical analysis were performed by two-tailed unpaired Student’s test (E-P). *p<0.05, **p < 0.01, ***p<0.001, ****p < 0.0001. Data are representative from ≥3 experiments (C,D,M) or pooled from ≥3 experiments (E-F,H-L,N-P) with each group containing 3 (C,D), 8-7 (E), 10 (F), 6-5 (H), 6 (I-J,L), 7 (K), or 7-6 (N-P) mice.

Article Snippet: The following list of antibodies and their concentration against mouse proteins were employed: αCD45 (1:400; BioLegend Cat# 103147, RRID:AB 2564383), αCD3ε (1:300; Thermo Fisher Scientific Cat# 46-0032-82, RRID:AB_1834427), αCD4 (1:300; BioLegend Cat# 100406, RRID:AB_312691), αCD8a (1:300; BD Biosciences Cat# 741811, RRID:AB_2871149), αCD11b (1:800; BD Biosciences Cat# 612801, RRID:AB_2870128), αLy6C (1:300; BD Biosciences Cat# 563011, RRID:AB_2737949), αLy6G (1:300; BioLegend Cat# 127624, RRID:AB_10640819), αCD11c(1:300; BD Biosciences Cat# 564080, RRID:AB_2738580), αSiglecF (1:300; BD Biosciences Cat# 746668, RRID:AB_2743940), αTNFα (1:300; (BioLegend Cat# 506305, RRID:AB_315426), αCD200R (1:300; BioLegend Cat# 123908, RRID:AB_2074080), αPD-L1 (1:300; BioLegend Cat# 124321, RRID:AB_2563635), αKi67 (1:300; (Thermo Fisher Scientific Cat# 56-5698-82, RRID:AB_2637480), αGM-CSFR (1:300; Thermo Fisher Scientific Cat# MA5-23918, RRID:AB_2608189), αGM-CSF(1:100; BioLegend Cat# 505406, RRID:AB_315382), αMHCII (1:300; BioLegend Cat# 107624, RRID:AB_2191073), αABCG1 (1:200; Bioss Cat# bs-1231R-FITC, RRID:AB_11120127), αEPCAM (1:300; BioLegend Cat# 118212, RRID:AB_1134101), αCD31 (1:300; MEC13.3, BioLegend Cat# 102424, RRID:AB_2650892) and αproSPC (1:300; Abcam Cat# ab270521).The following antibody and the concentration used against human proteins were employed: αEGFR (1:1000 for western blot; Cell Signaling Technology Cat# 4267, RRID:AB_2246311), αEGFR L858R mutant specific (1:100 microscopy; Cell Signaling Technology Cat# 3197, RRID:AB_1903955), αpEGFR (1:100; phospho Y1069) (Abcam Cat# ab205828, RRID:AB_2890267).

Techniques: Control, Flow Cytometry, Staining, Isolation, RNA Sequencing, Expressing, Liposomes, Enzyme-linked Immunosorbent Assay, Incubation, Blocking Assay, Two Tailed Test

Journal: bioRxiv

Article Title: EGFR + lung adenocarcinomas coopt alveolar macrophage metabolism and function to support EGFR signaling and growth

doi: 10.1101/2023.04.15.536974

Figure Lengend Snippet: (A) Bar graphs show SP-D concentrations in BALF collected from patients undergoing diagnostic lung lavages for lung cancer (n=5), COPD (n=3), or mycobacterial infection (n=2) as measured by ELISA. (B) Concentration of SP-D and SP-A from the BALF of littermate control (WT Heathy, black) mice or those with LUAD (WT Tumor, red) as measured by ELISA. (C) Bar graph shows the fold change in surfactant proteins mRNAs from EGFR L858R lung epithelium (EPCAM + ) in LUAD lungs . (D) SPA -/- SPD -/- double knockout mice were crossed with Ccsp-rtTA;TetO-EGFR L858R mice and placed on DOX for 6-8 weeks and dry lung weight was measured. (E-F) Similar to (A-B), heatmaps show lipids in the BALF from patients with lung cancer (red), COPD (black), or mycobacterial infection (gray) (E) or from littermate control (WT Heathy, black) mice or those with LUAD (WT Tumor, red) (F) as measured by LC/MS. Heatmaps depict the relative abundance of each class of lipid species normalized to volume (shown as a row Z score). Human data depict abundance averaged amongst the samples. (G-H) Import of free fatty acids, cholesterol and phospholipids (Bodipy C12, NBD cholesterol, DPPE respectively) were compared between AMs isolated littermate controls (H-AM, black) and LUAD mice (TA-AM, red) using flow cytometry and displayed as representative histograms of lipid import (G) and cumulative bar graphs of MFI (H). (I-K) H-AMs and TA-AMs were isolated 6-8 weeks on DOX and rates of basal mitochondrial respiration was measured using Seahorse Flux Analyzer or cells were labeled with 13 C-palmitate (J) or 13 C-glucose (K) and measured for rates of fatty acid oxidation and other metabolites, respectively. Data shown are mean ± SEM, and statistical analysis were performed with a two-tailed unpaired Students test (A-C, H-K) or a two way ANOVA (D). *p<0.05, **p < 0.01, ***p<0.001, ****p < 0.0001. Data are pooled from ≥3 experiments with each group containing 6 (B), 7-9 (D), 5-13 (H) animals or is representative from ≥2 experiments with each group containing 3 (G,I-K) animals. Human samples were collected from 3 (COPD), 2 (mycobacterial infection) or 5 (lung cancer) and data was pooled (A) or averaged (D).

Article Snippet: The following list of antibodies and their concentration against mouse proteins were employed: αCD45 (1:400; BioLegend Cat# 103147, RRID:AB 2564383), αCD3ε (1:300; Thermo Fisher Scientific Cat# 46-0032-82, RRID:AB_1834427), αCD4 (1:300; BioLegend Cat# 100406, RRID:AB_312691), αCD8a (1:300; BD Biosciences Cat# 741811, RRID:AB_2871149), αCD11b (1:800; BD Biosciences Cat# 612801, RRID:AB_2870128), αLy6C (1:300; BD Biosciences Cat# 563011, RRID:AB_2737949), αLy6G (1:300; BioLegend Cat# 127624, RRID:AB_10640819), αCD11c(1:300; BD Biosciences Cat# 564080, RRID:AB_2738580), αSiglecF (1:300; BD Biosciences Cat# 746668, RRID:AB_2743940), αTNFα (1:300; (BioLegend Cat# 506305, RRID:AB_315426), αCD200R (1:300; BioLegend Cat# 123908, RRID:AB_2074080), αPD-L1 (1:300; BioLegend Cat# 124321, RRID:AB_2563635), αKi67 (1:300; (Thermo Fisher Scientific Cat# 56-5698-82, RRID:AB_2637480), αGM-CSFR (1:300; Thermo Fisher Scientific Cat# MA5-23918, RRID:AB_2608189), αGM-CSF(1:100; BioLegend Cat# 505406, RRID:AB_315382), αMHCII (1:300; BioLegend Cat# 107624, RRID:AB_2191073), αABCG1 (1:200; Bioss Cat# bs-1231R-FITC, RRID:AB_11120127), αEPCAM (1:300; BioLegend Cat# 118212, RRID:AB_1134101), αCD31 (1:300; MEC13.3, BioLegend Cat# 102424, RRID:AB_2650892) and αproSPC (1:300; Abcam Cat# ab270521).The following antibody and the concentration used against human proteins were employed: αEGFR (1:1000 for western blot; Cell Signaling Technology Cat# 4267, RRID:AB_2246311), αEGFR L858R mutant specific (1:100 microscopy; Cell Signaling Technology Cat# 3197, RRID:AB_1903955), αpEGFR (1:100; phospho Y1069) (Abcam Cat# ab205828, RRID:AB_2890267).

Techniques: Diagnostic Assay, Infection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Control, Double Knockout, Liquid Chromatography with Mass Spectroscopy, Isolation, Flow Cytometry, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: EGFR + lung adenocarcinomas coopt alveolar macrophage metabolism and function to support EGFR signaling and growth

doi: 10.1101/2023.04.15.536974

Figure Lengend Snippet: (A) Violin plots of of PPARG mRNA in macrophage subsets from patients with NSCLC or matched healthy adjacent tissue (data from GSE131907). (B) MFI of PPARγ in various myeloid subsets in paired samples from NSCLC tumors or healthy adjacent tissue from the same patient measured by CYTOF analysis (data from Lavin et al. 2017). The following gating strategy was used: AMs: CD11b + CD64 + CD163 + CD206 hi , IMs: CD11b + CD64 + CD163 - , CD14 + Monocytes: CD11b + CD64 - CD14 + , and CD16 + Monocytes: CD11b + CD64 - CD16 + . (C-G) LUAD mice with deletion of PPARγ in macrophages ( Pparγ Fl/Fl ; Csf1r Cre , Ccsp-rtTA; TetO-EGFR L858R ) (referred to as PPARγ Tumors, blue) and littermate controls ( Ppar γ Fl/F ; Ccsp-rtTA; TetO-EGFR L858R ) (referred to as WT Tumors, red) were placed on DOX for 7-9 weeks at which point tumor burden and immune infiltrates were examined by scRNA-seq, microscopy and flow cytometry. Healthy lungs (black) were from control TetO-EGFR L858R mice on DOX. (C) Bar graph of dry lung weights. (D) Immunofluorescence microscopy was performed on lung sections from WT Tumor (red) and PPARγ -/- Tumor (blue) measuring density of tumor cells (EGFR L858R ), AMs (F4/80) and nuclei (dapi). (E) Heatmap shows Z-score by row of PPAR-target gene expression (from scRNA-seq) in the AM cluster from healthy (H, black) lungs or those with LUAD from WT (T, red) and PPARγ -/- (KO, blue) Tumors. (F-H) Percentage of AMs (F) and MFI of PD-L1 on AMs (G,H) as assessed by flow cytometry from the three groups of mice. (I-J) H-AMs (black) or TA-AMs from WT (red) and PPARγ -/- (blue) Tumors were isolated and stimulated with LPS to measure TNF production by flow cytometry. (K) Spheroids from human EGFR del19 cell line HCC827 were cultured on low attachment plates for a week and then AMs isolated from healthy lungs or those with LUAD from WT (red) or PPARγ -/- (blue) Tumors were added with GM-CSF (20 pg/mL) for three days and proliferation was measured by ki67 staining. (L-N) LUAD mice and littermate controls were placed on DOX for three weeks and then treated by oral gauvage 5x/week with the PPARγ antagonist GW9662 (1mg/kg) in corn oil or vehicle alone for three more weeks. (L) Representative MRI images and quantification of tumor burden in untreated and antagonist treated lungs. Percentage of AMs (M) and those producing TNF after LPS stimulation (N) were assessed by flow cytometry. Data shown are mean ± SEM, and statistical analysis were performed with a two-tailed unpaired Students test (D,L-N) or a two way ANOVA (C,F,H,J-K). *p<0.05, **p < 0.01, ***p<0.001, ****p < 0.0001. Data are pooled from ≥3 experiments with each group containing 13 (B) patients and 4 (D), 17-21 (F), 12 (H), 8-10 (J) 10-12 (K), 10-15 (L,M) and 10-14 (N) mice.

Article Snippet: The following list of antibodies and their concentration against mouse proteins were employed: αCD45 (1:400; BioLegend Cat# 103147, RRID:AB 2564383), αCD3ε (1:300; Thermo Fisher Scientific Cat# 46-0032-82, RRID:AB_1834427), αCD4 (1:300; BioLegend Cat# 100406, RRID:AB_312691), αCD8a (1:300; BD Biosciences Cat# 741811, RRID:AB_2871149), αCD11b (1:800; BD Biosciences Cat# 612801, RRID:AB_2870128), αLy6C (1:300; BD Biosciences Cat# 563011, RRID:AB_2737949), αLy6G (1:300; BioLegend Cat# 127624, RRID:AB_10640819), αCD11c(1:300; BD Biosciences Cat# 564080, RRID:AB_2738580), αSiglecF (1:300; BD Biosciences Cat# 746668, RRID:AB_2743940), αTNFα (1:300; (BioLegend Cat# 506305, RRID:AB_315426), αCD200R (1:300; BioLegend Cat# 123908, RRID:AB_2074080), αPD-L1 (1:300; BioLegend Cat# 124321, RRID:AB_2563635), αKi67 (1:300; (Thermo Fisher Scientific Cat# 56-5698-82, RRID:AB_2637480), αGM-CSFR (1:300; Thermo Fisher Scientific Cat# MA5-23918, RRID:AB_2608189), αGM-CSF(1:100; BioLegend Cat# 505406, RRID:AB_315382), αMHCII (1:300; BioLegend Cat# 107624, RRID:AB_2191073), αABCG1 (1:200; Bioss Cat# bs-1231R-FITC, RRID:AB_11120127), αEPCAM (1:300; BioLegend Cat# 118212, RRID:AB_1134101), αCD31 (1:300; MEC13.3, BioLegend Cat# 102424, RRID:AB_2650892) and αproSPC (1:300; Abcam Cat# ab270521).The following antibody and the concentration used against human proteins were employed: αEGFR (1:1000 for western blot; Cell Signaling Technology Cat# 4267, RRID:AB_2246311), αEGFR L858R mutant specific (1:100 microscopy; Cell Signaling Technology Cat# 3197, RRID:AB_1903955), αpEGFR (1:100; phospho Y1069) (Abcam Cat# ab205828, RRID:AB_2890267).

Techniques: Microscopy, Flow Cytometry, Control, Immunofluorescence, Targeted Gene Expression, Isolation, Cell Culture, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: EGFR + lung adenocarcinomas coopt alveolar macrophage metabolism and function to support EGFR signaling and growth

doi: 10.1101/2023.04.15.536974

Figure Lengend Snippet: LUAD mice lacking PPARγ in macrophages (PPAR -/- Tumors or KO Tumors (blue)) and littermate ‘WT Tumor’ controls ( Ppar γ Fl/F ; Ccsp-rtTA; TetO-EGFR L858R (red)) along with ‘WT Healthy’ lung controls ( TetO-EGFR L858R or Ccsp-rtTA (black)) were placed on DOX for 7-9 weeks. (A) Heatmap depicts the relative abundance (averaged across 3 samples/group) of the indicated lipid species in BALF normalized to the total volume as measured by LC/MS. (B) WT and PPARγ -/- KO TA-AMs were compared by scRNA-seq to identify the top 10 differentially upregulated pathways using GSEA. (C) Heatmap shows expression of fatty acid metabolism genes by row Z-score (from scRNA-seq) in WT AMs from Healthy lungs (black) vs. WT (red) and PPAR -/- (KO, blue) TA-AMs from LUAD lungs. (D-I) AMs isolated from WT Healthy lungs (black) and TA-AMs isolated WT (red) or PPARγ -/- (blue) Tumors were incubated with (D) [ 13 C]-palmitate, (E) [ 14 C]-acetate, (F) [ 14 C]-acetate, [ 14 C]-palmitate, or (I) [ 13 C]-glucose and assayed for (D) free fatty acid (FFA) import, (E) FA synthesis, (F) sterol synthesis, (G) FA b-oxidation (I) or relative flux into several metabolites. (H) Basal mitochondrial respiration in AMs was performed using seahorse extracellular flux assay. (J) Heatmap shows expression of cholesterol synthesis and metabolism genes by row Z-score (from scRNA-seq) in WT AMs from Healthy lungs (black) vs. WT (red) and PPARγ -/- (KO, blue) TA-AMs from LUAD lungs. (K) ABCG1 expression on AMs was assessed by flow cytometry. (L-M) Control and PPARγ -/- TA-AMs were cultured with NBD-Cholesterol (green) for one hour and then (L) imaged by confocal microscopy to examine lipid droplets. To assess cholesterol efflux (M), the labeled TA-AMs and H-AMs were then equilibrated overnight in serum free media followed by incubation with FBS as a cholesterol acceptor for 4 hours and effluxed NBD-Cholesterol in the supernatant was measured by fluorescence. Data shown are mean ± SEM, and statistical analysis were performed with a two way ANOVA. *p<0.05, **p < 0.01, ***p<0.001, ****p < 0.0001. Data are representative from 2 experiments with an N=3 (A). Data are pooled from ≥2 experiments with each group containing 6 (D), 5 (E, F), 3 (G-H), 5-6 (I) or 5-11 (M) animals.

Article Snippet: The following list of antibodies and their concentration against mouse proteins were employed: αCD45 (1:400; BioLegend Cat# 103147, RRID:AB 2564383), αCD3ε (1:300; Thermo Fisher Scientific Cat# 46-0032-82, RRID:AB_1834427), αCD4 (1:300; BioLegend Cat# 100406, RRID:AB_312691), αCD8a (1:300; BD Biosciences Cat# 741811, RRID:AB_2871149), αCD11b (1:800; BD Biosciences Cat# 612801, RRID:AB_2870128), αLy6C (1:300; BD Biosciences Cat# 563011, RRID:AB_2737949), αLy6G (1:300; BioLegend Cat# 127624, RRID:AB_10640819), αCD11c(1:300; BD Biosciences Cat# 564080, RRID:AB_2738580), αSiglecF (1:300; BD Biosciences Cat# 746668, RRID:AB_2743940), αTNFα (1:300; (BioLegend Cat# 506305, RRID:AB_315426), αCD200R (1:300; BioLegend Cat# 123908, RRID:AB_2074080), αPD-L1 (1:300; BioLegend Cat# 124321, RRID:AB_2563635), αKi67 (1:300; (Thermo Fisher Scientific Cat# 56-5698-82, RRID:AB_2637480), αGM-CSFR (1:300; Thermo Fisher Scientific Cat# MA5-23918, RRID:AB_2608189), αGM-CSF(1:100; BioLegend Cat# 505406, RRID:AB_315382), αMHCII (1:300; BioLegend Cat# 107624, RRID:AB_2191073), αABCG1 (1:200; Bioss Cat# bs-1231R-FITC, RRID:AB_11120127), αEPCAM (1:300; BioLegend Cat# 118212, RRID:AB_1134101), αCD31 (1:300; MEC13.3, BioLegend Cat# 102424, RRID:AB_2650892) and αproSPC (1:300; Abcam Cat# ab270521).The following antibody and the concentration used against human proteins were employed: αEGFR (1:1000 for western blot; Cell Signaling Technology Cat# 4267, RRID:AB_2246311), αEGFR L858R mutant specific (1:100 microscopy; Cell Signaling Technology Cat# 3197, RRID:AB_1903955), αpEGFR (1:100; phospho Y1069) (Abcam Cat# ab205828, RRID:AB_2890267).

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing, Isolation, Incubation, XF Assay, Flow Cytometry, Control, Cell Culture, Confocal Microscopy, Labeling, Fluorescence

Journal: bioRxiv

Article Title: EGFR + lung adenocarcinomas coopt alveolar macrophage metabolism and function to support EGFR signaling and growth

doi: 10.1101/2023.04.15.536974

Figure Lengend Snippet: (A) Heatmap shows expression of EGFR signaling genes by row Z-score (from scRNA-seq) in AT2 cells isolated from healthy lungs (black) or LUAD lungs that contain WT (red) or PPARγ TA-AMs (blue). (B-C) Lungs from same samples as in (A) were stained with mAbs to phospho-EGFR (pEGFR) and the AT2 cell marker pro-SPC and analyzed by confocal microscopy (B). Data are representative of sections taken from four separate mice. The intensity of pEGFR staining in AT2 was quantified using IMARIS (C). (D-E) The EGFR Del19 mutant human cell line HCC827 was cultured for 1-3 days in the absence or presence of 10 μM atorvastatin and each day the viable cell number was enumerated (D) and the amounts of pEGFR relative to total EGFR were assessed by Western blotting (E). (F-K) LUAD lungs containing WT (red) or PPARγ (blue) were treated by oral gavauge with pravastatin (0.5 mg, 5X/week) from weeks 3-7 on DOX. Tumor burden was measured using confocal microscopy staining with mAbs specific for the EGFR L858R oncogene, macrophage marker F4/80, and ki67 (F, G) and by dry lung weight (H). (I-K) Intracellular cytokine staining, and flow cytometry was used to assess the frequency of TNF-producing AMs (I), CD4 + and CD8 + T cells (J, K) after stimulation with LPS (I) or PMA and ionomycin (J, K). Data shown are mean ± SEM, and statistical analysis were performed with a two-tailed unpaired Students test (E) or a two-way ANOVA (C,G,I-M). *p<0.05, **p < 0.01, ***p<0.001, ****p < 0.0001. Data are representative of ≥3 mice (B,C,F) and is pooled from ≥3 experiments with each group containing 50-149 cells (C) and 8 (D), 6-9 (G), 5-6 (H), 5-7 (I), 3-6 (J-K) mice.

Article Snippet: The following list of antibodies and their concentration against mouse proteins were employed: αCD45 (1:400; BioLegend Cat# 103147, RRID:AB 2564383), αCD3ε (1:300; Thermo Fisher Scientific Cat# 46-0032-82, RRID:AB_1834427), αCD4 (1:300; BioLegend Cat# 100406, RRID:AB_312691), αCD8a (1:300; BD Biosciences Cat# 741811, RRID:AB_2871149), αCD11b (1:800; BD Biosciences Cat# 612801, RRID:AB_2870128), αLy6C (1:300; BD Biosciences Cat# 563011, RRID:AB_2737949), αLy6G (1:300; BioLegend Cat# 127624, RRID:AB_10640819), αCD11c(1:300; BD Biosciences Cat# 564080, RRID:AB_2738580), αSiglecF (1:300; BD Biosciences Cat# 746668, RRID:AB_2743940), αTNFα (1:300; (BioLegend Cat# 506305, RRID:AB_315426), αCD200R (1:300; BioLegend Cat# 123908, RRID:AB_2074080), αPD-L1 (1:300; BioLegend Cat# 124321, RRID:AB_2563635), αKi67 (1:300; (Thermo Fisher Scientific Cat# 56-5698-82, RRID:AB_2637480), αGM-CSFR (1:300; Thermo Fisher Scientific Cat# MA5-23918, RRID:AB_2608189), αGM-CSF(1:100; BioLegend Cat# 505406, RRID:AB_315382), αMHCII (1:300; BioLegend Cat# 107624, RRID:AB_2191073), αABCG1 (1:200; Bioss Cat# bs-1231R-FITC, RRID:AB_11120127), αEPCAM (1:300; BioLegend Cat# 118212, RRID:AB_1134101), αCD31 (1:300; MEC13.3, BioLegend Cat# 102424, RRID:AB_2650892) and αproSPC (1:300; Abcam Cat# ab270521).The following antibody and the concentration used against human proteins were employed: αEGFR (1:1000 for western blot; Cell Signaling Technology Cat# 4267, RRID:AB_2246311), αEGFR L858R mutant specific (1:100 microscopy; Cell Signaling Technology Cat# 3197, RRID:AB_1903955), αpEGFR (1:100; phospho Y1069) (Abcam Cat# ab205828, RRID:AB_2890267).

Techniques: Expressing, Isolation, Staining, Marker, Confocal Microscopy, Mutagenesis, Cell Culture, Western Blot, Flow Cytometry, Two Tailed Test