Journal: Cancer immunology research

Article Title: Antitumor effects of CAR T cells redirected to the EDB splice variant of fibronectin

doi: 10.1158/2326-6066.CIR-20-0280

Figure Lengend Snippet: (A) Scheme of retroviral vectors encoding the EDB-CAR, a 2A sequence, and truncated CD19 (tCD19); SH: short hinge; TM: transmembrane domain. (B-C) EDB-CAR expression was determined on T cells 7 days post-transduction by flow cytometric analysis. (B) Representative histogram (Black-filled line: NT T cells; Red-filled line: EDB-CAR transduced T cells) and (C) summary plot (n=7; Student’s t-test; ****p<0.0001). (D) NT and EDB-CAR T cells were incubated for 24 hours with increasing concentrations of rhFN-EDB-coated wells. IFNγ production in supernatants was determined by ELISA (n=4; two-way ANOVA; ***p<0.001; ****p<0.0001). (E) EDB expression of LM7, A673, A549, and U87 tumor cells, and two primary fibroblast cell lines (Fib 1, Fib 2) determined by RT-qPCR; dotted line represents the ΔCt score above which samples are considered positive. (F) NT and EDB-CAR T cells were incubated for 48 hours with EDB-positive tumor cells. IFNγ and IL2 production in supernatants was determined by ELISA (two-way ANOVA; ****p<0.0001). (G) Cytolytic activity of NT or EDB-CAR T cells at a E:T ratio of 4:1 against EDB-positive tumor cells (MTS assay; n=3; two-way ANOVA; *p<0.05; **p<0.01; ***p<0.001). (H) Cytolytic activity of NT or EDB-CAR T cells against primary fibroblasts (data of Fib 1 and Fib 2 was combined) at the indicated E:T ratios, with A549 serving as controls (n=3; two-way ANOVA; ****p<0.0001). (I) NT, EDB-CAR, or mutEDB-CAR T cells were incubated for 48 hours with U87 or U87FN−/− cells. IFNγ production in supernatants was determined by ELISA (n=3; two-way ANOVA; ****p<0.0001). (J) Cytolytic activity of NT, EDB-CAR, or mutEDB-CAR T cells against U87 or U87FN−/− cells (n=3; two-way ANOVA; ****p<0.0001). Mean+SEM is shown in panels.

Article Snippet: Experiments with CD31-positive vascular endothelial cells Mice were injected s.c. with 2×10 6 U87 or A673 tumor cells in Matrigel (Corning; 1:1 diluted in PBS).

Techniques: Retroviral, Sequencing, Expressing, Transduction, Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Activity Assay, MTS Assay

Journal: Cancer immunology research

Article Title: Antitumor effects of CAR T cells redirected to the EDB splice variant of fibronectin

doi: 10.1158/2326-6066.CIR-20-0280

Figure Lengend Snippet: (A-H) 2×106 of the indicated tumor cells were injected into NSG mice s.c.. On day (D)7 (U87, A673, U87FN−/−) or D10 (A549) post tumor cell injection, mice received a single i.v. dose of 1×106 NT or EDB-CAR T cells. Tumor growth was monitored weekly by caliper measurement (n=10 mice/group; 5 mice/T-cell donor; log-rank test; *p<0.05; ***p<0.001). (A) Experimental scheme and tumor growth for the U87 model. (B) U87 model: Kaplan-Meier survival curve. (C) Experimental scheme and tumor growth for the A549 model. (D) A549 model: Kaplan-Meier survival curve. (E) Experimental scheme and tumor growth for the A673 model. (F) A673 model: Kaplan-Meier survival curve. (G) Experimental scheme and tumor growth for the U87FN−/− model. (H) U87FN−/− model: Kaplan-Meier survival curve. (I) NSG mice that survived long-term post initial A549 (n=2) or U87 (n=3) s.c. tumor cell injection and EDB-CAR T-cell therapy were re-challenged with a single s.c. dose of cognate tumor cells. Naïve mice served as controls. Tumor growth was monitored weekly by caliper measurement. (J) Macroscopic tumor necrosis of tumors <2,000 mm3 (Student’s t-test; *p<0.05) of the experiments shown in panels A-I; dots represent how many tumors had macroscopic necrosis out of 10 tumors per cell type; dotted line represent baseline (1 tumor with macroscopic necrosis).

Article Snippet: Experiments with CD31-positive vascular endothelial cells Mice were injected s.c. with 2×10 6 U87 or A673 tumor cells in Matrigel (Corning; 1:1 diluted in PBS).

Techniques: Injection

Journal: Cancer immunology research

Article Title: Antitumor effects of CAR T cells redirected to the EDB splice variant of fibronectin

doi: 10.1158/2326-6066.CIR-20-0280

Figure Lengend Snippet: (A-D) A673 or A549 s.c. tumor-bearing mice received a single dose of NT, EDB-CAR, or EphA2-CAR (A549 only) T cells. Tumors were processed on day (D)10 (A673) or 14 (A549) post T-cell injection and analyzed for CD31 expression (n=4 mice per group with each mouse bearing one tumor; dots represent average of three blind scores per tumor; bars represent average per cohort, two fields-of-view (FOV) per tumor). (B) CD31+ vessels/FOV in the A673 model; Student’s t-test; **p<0.01. (C) CD31+ vessels/FOV in the A549 model; one-way ANOVA; *p<0.05). (D) Representative IHC images for CD31 staining of A549 tumors. Images at 40x magnification; scale bar: 100μm. (E-G) A673 s.c. tumor-bearing NSG mice were imaged using Angiosense 750 on day −1 and day 14 post NT or EDB-CAR T-cell injection. (E) Experimental scheme. (F) Representative images. (G) Quantitation (relative light units (RLU) divided by tumor volume is plotted; n=5 mice/treatment group; Student’s t-test; *p<0.05). (H) Experimental scheme. s.c. tumor-bearing (I) U87 or (J) U87FN−/− NSG mice received a single i.v. dose of NT or EDB-CAR T cells. Tumor were processed on day 10 post T-cell injection and analyzed for CD31 expression (n=4 mouse/tumors per group except for U87FN−/−/NT T-cell group (n=3) with dots representing three blind score per FOV; bars represent average per cohort, 2 FOV per tumor). (K) Representative IHC images for CD31 for U87 and U87FN−/− cells. Images at 40x magnification; scale bar: 100μm.

Article Snippet: Experiments with CD31-positive vascular endothelial cells Mice were injected s.c. with 2×10 6 U87 or A673 tumor cells in Matrigel (Corning; 1:1 diluted in PBS).

Techniques: Injection, Expressing, Staining, Quantitation Assay

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Integrated bioinformatics analysis of miRNA expression in Ewing sarcoma and potential regulatory effects of miR-21 via targeting ALCAM/CD166.

doi: 10.1080/21691401.2019.1620760

Figure Lengend Snippet: Figure 11. (A) Schematic diagram of luciferase reporter assay. The green dots represent stronger fluorescence signal, the blue dots represent weak fluorescence sig- nal. (B) Relative luciferase activity of indicated ALCAM reporter constructs in 293 T cells. Transfection of miR-21 suppressed the activity of the luciferase reporter which contained the 30-UTR of ALCAM significantly but did not influence the luciferase activity of the mutated 30-UTR structure in 293 T cells. p < .01. (C) Microscopic observation of hMSC cell line, A673 cell line and 293 T cells.

Article Snippet: Cell lines and cell culture The human Ewing sarcoma cell lines A673 SK-ES-1 RD-ES and the hMSC cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Luciferase, Reporter Assay, Fluorescence, Activity Assay, Construct, Transfection

Journal: bioRxiv

Article Title: Trabectedin Enhances Oncolytic Virotherapy by Reducing Barriers to Virus Spread and Cytotoxic Immunity in Preclinical Pediatric Bone Sarcoma

doi: 10.1101/2024.03.02.582994

Figure Lengend Snippet: oHSV+trabectedin synergizes to increase the disease control rate and reduce tumor burden in human xenograft models. The best response for each treated tumor through 28 days, the average tumor burden, and spider plots tracking individual tumor volumes over the full study period are shown for A) CHLA-258 (Ewing sarcoma), B) EW5 (Ewing sarcoma), C) PDX-0027 (rhabdomyosarcoma), D) A673 (Ewing sarcoma), and E) A673 in NSG-SGM3 NK-deficient mice (lack T, B, and NK cells). PBS and oHSV (1.0 x 107 pfu) were given i.Tu. on Days 0 & 2. Trabectedin (0.15 mg/kg) was given I.V. on Days 0 & 7. Statistical analyses of the disease control rates (CR+PR+SD) were performed using a pairwise Fisher’s exact test with p values adjusted using the Benjamini-Hochberg procedure; *p < 0.05, **p < 0.01, ***p < 0.001. Summarized data with error bars depict mean ± SEM.

Article Snippet: The A673 human Ewing sarcoma cell line (Cat# CCL-81), Vero green monkey kidney cell line (Cat# CRL-1598), and K7M2 mouse osteosarcoma cell line (Cat# CRL-2836) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Control

Journal: bioRxiv

Article Title: Trabectedin Enhances Oncolytic Virotherapy by Reducing Barriers to Virus Spread and Cytotoxic Immunity in Preclinical Pediatric Bone Sarcoma

doi: 10.1101/2024.03.02.582994

Figure Lengend Snippet: Trabectedin increases viral spread and decreases antiviral gene expression in immunodeficient Ewing sarcoma models. A) Treatment schema for scRNAseq tumor collection: PBS and oHSV (1.0 x 10 pfu) were given i.Tu. on Days 0 & 2, trabectedin (0.15 mg/kg) was given I.V. on Day 0, and scRNAseq samples were collected on Day 3 to ensure sufficient cell viability for sequencing. B) oHSV viral titer (pfu) from A673 tumors treated with a single dose of oHSV or oHSV+trabectedin. C) UMAP plots of oHSV transcript presence in all tumor cells, split by treatment group. D) Violin plot of the expression level (log-transformed) of all oHSV transcripts for all tumor cells in each treatment group. E) Expression level of HSV time-dependent gene modules shown as a feature plot (UMAP) for all tumor cells and split by treatment groups that contained oHSV. A relative expression cutoff of 1 minimized color skewing by high expressing outlier cells and maintained color scale consistency between feature plots. F) Violin plot of the expression level (log-transformed) of HSV time-dependent gene modules for all tumor cells in treatment groups that contained oHSV. G) Results from gene set enrichment analysis showing the running enrichment score for the “Herpes simplex virus 1 infection” pathway, which represents the intrinsic cellular response to HSV-1. Gene set enrichment analysis was performed on a gene list which was ranked by the gene expression fold change (log2FC) calculated for tumor cells treated with oHSV+trabectedin compared to those treated with oHSV monotherapy. The schema displays the top genes with enrichment changes from the “Herpes simplex virus 1 infection” pathway.

Article Snippet: The A673 human Ewing sarcoma cell line (Cat# CCL-81), Vero green monkey kidney cell line (Cat# CRL-1598), and K7M2 mouse osteosarcoma cell line (Cat# CRL-2836) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Gene Expression, Sequencing, Expressing, Transformation Assay, Virus, Infection