tnf α  (R&D Systems)

Journal: Cancer Research

Article Title: Human 3D Ovarian Cancer Models Reveal Malignant Cell–Intrinsic and –Extrinsic Factors That Influence CAR T-cell Activity

doi: 10.1158/0008-5472.CAN-23-3007

Figure Lengend Snippet: Impaired death receptor signaling in malignant cells caused resistance to CAR T-cell cytotoxicity. A, Hierarchical cluster analysis of transcriptomes for OvCAR3 and G164 cells in monolayer and spheroids. B, Heatmap illustrating normalized gene expression of differentially expressed genes relating to death receptor signaling in OvCAR3 and G164 cells (adjusted P value < 0.05). C, cIAP1/2 expression on human HGSOC omental metastasis ( n = 16) and adjacent omentum ( n = 10). Statistics performed using two-way ANOVA. D and E, Representative images (left) and quantification (right) of Incucyte killing assay in which monolayers of G164 ( D ) and G33 ( E ) cells were treated with birinapant and CAR T cells. F, Western blot showing the expression of cIAP2 in OvCAR3 and G164 cells treated with birinapant. Representative images of three repeats. G, ELISA data showing TNFα concentration after coculturing CAR T cells with monolayers of OvCAR3 and G164 cells for 2 days at 1:5 T:E ratios. Data plotted as mean ± SD for three CAR T-cell donors. Statistics performed using two-way ANOVA. H, Representative images (left) and quantification (right) of Incucyte killing assay in which OvCAR3 monolayer was treated with anti-TNFα antibody and CAR T cells. D–F, Data shown for one CAR T-cell donor at 1:5 T:E ratio. Images shown are 3 days after treatment. Red, dead cells. Scale bars, 400 μm.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) was performed using human IFNα Quantikine kit (R&D Systems, Cat. DIF50C), human TNFα QuantiGlo kit (R&D Systems, Cat. QTA00C), human CCL2/MCP1 Quantikine kit (R&D Systems, Cat. DCP00), and human TGFβ1 Quantikine kit (R&D Systems, Cat. DB100C) according to manufacturer’s instructions.

Techniques: Gene Expression, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Cancer Research

Article Title: Human 3D Ovarian Cancer Models Reveal Malignant Cell–Intrinsic and –Extrinsic Factors That Influence CAR T-cell Activity

doi: 10.1158/0008-5472.CAN-23-3007

Figure Lengend Snippet: Vascularized microfluidic chip to investigate CAR T-cell migration and cytotoxicity. A, Design of the tri-channel microfluidic device with a 2 mm well in the central channel. B, Schematic diagram showing the development of ovarian cancer-on-a-chip model. Scale bar, 600 μm. C, Immunofluorescence image showing microvasculature in fibrin and OvCAR3 collagen gels ( n = 3). Red, HUVEC. Scale bar, 100 μm. D, Real-time images showing the luminal flow of CAR T cells (arrows) through the vasculature formed within the microfluidic device ( n = 2). Green, CAR T cells. Scale bar, 20 μm. E, Immunofluorescence image showing an ovarian cancer-on-a-chip 3 days after CAR T-cell treatment. CAR T cells (arrows) migrated into the OvCAR3 gel in the middle of the device. Dotted line marks the edge of the central well. Green, CAR T cells; red, HUVEC. Scale bar, 100 μm. F and G, CD3 ( F ) and caspase‐3 (Casp3; G ) staining and quantification of OvCAR3 gels isolated from microfluidic device after CAR T-cell and anti-TNFα treatment. H, MSD data showing TNFα, IFNγ, and IL2 concentrations on media from microfluidic devices after CAR T-cell and anti-TNFα treatment. F–H, Data plotted as mean ± SD of two/three gels per two replicates. Two different CAR T-cell donors were used in this experiment. Scale bar, 50 μm. Statistics performed using two-way ANOVA.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) was performed using human IFNα Quantikine kit (R&D Systems, Cat. DIF50C), human TNFα QuantiGlo kit (R&D Systems, Cat. QTA00C), human CCL2/MCP1 Quantikine kit (R&D Systems, Cat. DCP00), and human TGFβ1 Quantikine kit (R&D Systems, Cat. DB100C) according to manufacturer’s instructions.

Techniques: Migration, Immunofluorescence, Staining, Isolation