Journal: Nature Communications

Article Title: In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo

doi: 10.1038/s41467-020-19486-2

Figure Lengend Snippet: B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Ai reporter) mice were injected intravenously with three daily doses of nanoparticles loaded with 15 μg mRNA encoding nuclear localization signal (NLS)-Cre. Nanoparticles were targeted to mouse T cells using a full-length anti-CD3 MuIgG2a, or IgG2a isotype control. Both antibodies were designed as LALAPG variants to ablate Fc receptor binding and complement activation. Forty-eight hours after the final injection, organs were collected and whole-organ dtTomato fluorescence was measured using fluorescent IVIS imaging. Single cell suspensions of spleens and blood were labeled with antibodies against various immune cell subtypes and analyzed by flow cytometry. a Representative ( N = 7, 1 pictured) dtTomato expression in organs under fluorescent IVIS imaging. b Quantification of fluorescent signal in each organ. Each symbol indicates one measured organ. Horizontal lines indicate mean values, and error bars represent standard deviation of the mean. Pairwise differences in fluorescent intensities between the groups were statistically analyzed by two tailed unpaired Student’s t -test. N = 7 biologically independent samples pooled from two independent experiments. c Graph displaying the mean ± SD percent of immune CD45+ dtTomato+ cell types in the spleen. Macrophages (CD45+, CD11b+, MHCII+, CD11c−, Ly6C−/low, Ly6G−), B cells (CD45+, B220+), T cells [CD4+ T cells (CD45+, TCR-β chain+, CD4+, CD8-), CD8+ T cells (CD45+, TCR-β chain+, CD4−, CD8+)], neutrophils (CD45+, CD11b+, MHCII+, CD11c−, Ly6G+), and dendritic cells (CD45+, CD11c+, CD11b−, MHCII+) were measured. Each symbol indicates one mouse. Horizontal lines indicate mean values, and error bars represent standard deviation of the mean. Pairwise differences in fluorescent intensities between the groups were statistically analyzed by two tailed unpaired Student’s t -test. N = 7 biologically independent samples.

Article Snippet: Human CD45 (clone HI130, dilution 1:400, PE-Cyanine 7, ThermoFisher Cat#25-0459-42).

Techniques: Injection, Control, Binding Assay, Activation Assay, Fluorescence, Imaging, Labeling, Flow Cytometry, Expressing, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo

doi: 10.1038/s41467-020-19486-2

Figure Lengend Snippet: a Heat map of PSCA, PSMA, and ROR1 antigen expression across a panel of 140 prostate cancer metastases showing the diversity of antigen expression. b Heat map representation of flow cytometry data showing variability in PSCA, PSMA, and ROR1 expression by LNCap C42 prostate carcinoma cells. The colors indicate expression levels in 350 randomly chosen cells. c Three weeks of postimplantation, LNCap C42 prostate tumors were visualized by in vivo bioluminescent imaging. A representative photo of established tumors in the dorsal lobes of the prostates (white arrows) is shown on the right. d Sequential bioimaging of firefly luciferase-expressing LNCap C42 prostate carcinoma cells orthotopically transplanted into the prostate of NGS mice. Four representative mice from each cohort ( n = 8) are shown. e Time line and nanoparticle dosing regimen. f Survival of animals following therapy, depicted as Kaplan–Meier curves. Shown are eight mice per treatment group pooled from three independent experiments. ms, median survival. Statistical analysis between the treated experimental and the untreated control group was performed using the Log-rank test; P < 0.05 was considered significant. N.s. nonsignificant. g Multicolor flow cytometry of cells recovered from prostate tumors 11 days after treatment start. Adoptively transferred or in situ-programmed ROR1 CAR+ T cells were identified by positive labeling for CD45 and a c-myc tag incorporated in the receptor. h Absolute numbers of ROR1-CAR+ T cells that localized to tumors isolated on day 4, day 7, and day 11 after treatment start. Total cell counts of viable (trypan blue-negative) cells were multiplied by the percentage that was both RO1-CAR and CD45 positive. Shown are mean values ± SD; two tailed unpaired Student’s t -test. N = 8 biologically independent samples pooled from two independent experiments. i Flow cytometry quantification of ROR1 antigen expression on LNCaP C42 prostate tumor cells following CAR-T cell therapy or ROR1 4-1BBz CAR NP therapy. Shown are 350 randomly chosen cells pooled from five tumors.

Article Snippet: Human CD45 (clone HI130, dilution 1:400, PE-Cyanine 7, ThermoFisher Cat#25-0459-42).

Techniques: Expressing, Flow Cytometry, In Vivo, Imaging, Luciferase, Control, In Situ, Labeling, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo

doi: 10.1038/s41467-020-19486-2

Figure Lengend Snippet: a We established a mouse xenograft tumor model of HBV-induced HCC. HepG2 cells stably transduced with HBcAg and luciferase were surgically injected into the liver of NSG mice reconstituted with human T cells. Three weeks post-implantation, HepG2 tumors were visualized by in vivo bioluminescent imaging and assigned to nanoparticle (6 weekly doses of 50 μg mRNA encoding the HBcore18-17 TCR) or T-cell treatment (5 × 10 6 T cells transduced ex vivo with lentiviral vectors encoding the HBcore18-17 TCR) groups. b , c Quantification of bioluminescent liver signal 6 weeks after treatment start. Shown are mean values ± SD; two tailed unpaired Student’s t -test. N = 5 biologically independent samples. d Multicolor flow cytometry of cells recovered from the liver 18 days after treatment start. Adoptively transferred or in situ-programmed HBcore18-27 TCR+ T cells were identified by positive labeling for CD45, CD8, and MHC Pentamer. Absolute numbers are shown in e . Total cell counts of viable (trypan blue-negative) cells were multiplied by the percentage that was HBcore18–27 TCR+, CD8+, and CD45+. Shown are mean values ± SD. Pairwise differences in absolute numbers of T cells between the groups were statistically analyzed by two tailed unpaired Student’s t -test. P < 0.05 was considered significant. N.s. nonsignificant. N = 5 biologically independent samples.

Article Snippet: Human CD45 (clone HI130, dilution 1:400, PE-Cyanine 7, ThermoFisher Cat#25-0459-42).

Techniques: Stable Transfection, Transduction, Luciferase, Injection, In Vivo, Imaging, Ex Vivo, Two Tailed Test, Flow Cytometry, In Situ, Labeling

Journal: Cell Reports Medicine

Article Title: Cancer-associated fibroblasts undergoing neoadjuvant chemotherapy suppress rectal cancer revealed by single-cell and spatial transcriptomics

doi: 10.1016/j.xcrm.2023.101231

Figure Lengend Snippet:

Article Snippet: APC-Cyanine7 Anti-Human CD45 , Tonbo Biosciences , Cat# 25-0459-T100; RRID: AB_2621631.

Techniques: Recombinant, Red Blood Cell Lysis, Isolation, Saline, Expressing, Software, Reverse Transcription

Journal: Nature Communications

Article Title: Leukemic stem cells activate lineage inappropriate signalling pathways to promote their growth

doi: 10.1038/s41467-024-45691-4

Figure Lengend Snippet: A – C t(8;21) PDX cells were grown in vitro for 6 days with or without IL-5 and/or VEGF(165) ( A ), with 3 doses of bevacizumab ( B ) or with 3 doses of benralizumab (+10 ng/ml IL-5) ( C ) and the resulting cells counted. Control/0 bevacizumab sample in ( A ) and ( B ) is the same as experiments were performed in parallel. Bar height shows the mean of 3 wells and error bars indicate SEM, p = 0.0159 + IL-5, p = 0.0090 + VEGF in ( A ) and p = 0.0214 20 pg/ml, p = 0.0057 100 pg/ml, p = 0.0015 500 pg/ml benralizumab in ( B , C ). D Schematic showing how PDX dosing and sampling were conducted in vivo, analyses were performed on mice reaching day 90+. All mice taken prior to the fixed end point had weight loss or leg tumours associated with the PDX except * which had an enlarged thymus. E Representative contour plots showing the human and mouse CD45 positive cells by flow cytometry in peripheral blood at day 92 post-injection. F Percentage of human CD45 positive cells in peripheral blood at end point indicated in ( D ), normalised to the mean of the vehicle control mice for experiment 1 (CV1-3, + benralizumab (Ben) 1-3, + bevacizumab (Bev) 1-3) and experiment 2 (CV7-10, Ben6-9, Bev5-9), n = 7 mice for control, 7 mice benralizumab, 8 mice bevazicumab. P = 0.0997 benralizumab, p = 0.0035 bevacizumab. G , H Representative contour plots showing the relative populations of hCD45 + CD34 + CD38 + /− cells ( G ) and KDR and IL5RA positivity of hCD45+/CD34+/CD38+ blast cells and hCD45+/CD34+/CD38- LSCs ( H ) in control left bone marrow at day 92 post-injection. I Representative contour plots showing the KDR and IL5RA positivity of hCD45+/CD34+/CD38− LSCs in treated or control left bone-marrow at day 92 post-injection. J Percentage of KDR and IL5RA positive hCD45+/CD34+/CD38− LSCs in left bone marrow at day 92/99 post-injection, p = 0.0359 benralizumab, p = 0.0320 bevacizumab, n = 6 mice for control, 6 mice benralizumab, 7 mice bevacizumab. F , J Horizontal and error bars show mean and SEM respectively of the mice in each treatment group, * p < 0.05, ** p < 0.01, *** p < 0.005 using two-tailed unpaired Student’s t -tests vs control ( A ), one-way ANOVA with Bonferroni’s multiple comparisons ( B , C ) and two-tailed unpaired Welch’s t -test vs vehicle controls ( F , J ). Blue corresponds to bevacizumab and orange to benralizumab throughout. Source data are provided as a Source Data file.

Article Snippet: Flow cytometry was carried out on a Cyan ADP (Beckman Coulter) using antibodies against CD309-APC (KDR, Cat# 130-117-984 Miltenyi Biotec) and CD125-biotin (IL5RA, Cat# 130-110-543 Miltenyi Biotec) followed by streptavidin-PE-Cy7 (Cat# 25-4317-82 ThermoFisher) for cell lines, and on an Attune NxT (Thermo Fisher) using antibodies against 1: hCD45-FITC, CD34-APC Cat# 130-120-519, CD38-V450 Cat# 646851 BD Biosciences, VEGFR-APC-vio770 Cat# 130-117-987 and IL5RA-PE Cat# 130-110-602, 2: hCD45 APC-eFluor780 Cat# 47-0459-42 ThermoFisher, CD34-PE Cat# 130-120-515 and mCD45-APC, or 3: CD33-BV421, CD11b-APC Cat# 130-091-241, CD34-PE and hCD45-APC-eFluor780 for PDX cells.

Techniques: In Vitro, Sampling, In Vivo, Flow Cytometry, Injection, Two Tailed Test