Journal: Cell reports

Article Title: Different chromatin-scanning modes lead to targeting of compacted chromatin by pioneer factors FOXA1 and SOX2.

doi: 10.1016/j.celrep.2023.112748

Figure Lengend Snippet: Figure 3. While displaying opposite kinetics, FOXA1 and SOX2 both perform a dense scan- ning of compact regions (A) Simulating chromatin-scanning trajectories for 1,000 steps: after one step of nucleoplasmic diffu- sion (green), the probability to diffuse again (Pdiffusion, green) or interact with chromatin (Pinteraction, red) is inferred from diffusion coefficients. If chromatin interaction occurs, the probability to interact with a site in low- (Pcompact, black) versus high-mobility (Popen, orange) chromatin is inferred from radius of confinements and average displacements. (B–D) Representative scanning trajectories by a single molecule of FOXA1 (B), SOX2 (C), and HNF4A (D) exploring 1,000 chromatin sites. Each step of diffusion is set to occur in a random direction. Red dots: binding to low-mobility, compact chromatin. White dots: binding to high-mobility, open chro- matin. (E and F) Total time of chromatin interaction (E) or nucleoplasmic diffusion (F) during the exploration of 1,000 sites. (G) Average areas (mm2) after Delaunay triangulation of low-mobility chromatin interaction spatial co- ordinates for 10,000 simulated scanning trajectories. For all panels, ***p < 0.0001, n.s., non-significant differences (p > 0.05) as determined by one-way ANOVA.

Article Snippet: Gel extraction kit QIAGEN 28706 Gibson Assembly kit NEB E5510 NEBNext UltraTM II for DNA Library Prep NEB E7645 CUTANATM pAG-MNase Epicypher 15–1016 Deposited data FOXA1-HALO ChIP-seq This paper GSE220570 HNF4A-HALO ChIP-seq This paper GSE220570 H2B-HALO 0h CHIP-seq This paper GSE220570 H2B-HALO 6h ChIP-seq This paper GSE220570 MNase-seq This paper GSE220570 SOX2-DBD-V5 CUT&RUN This paper GSE220570 FOXA1-NHAA-HALO ChIP-seq This paper GSE220570 FOXA1-RRAA-HALO ChIP-seq This paper GSE220570 FOXA1-DBD-HALO ChIP-seq This paper GSE220570 SOX2-V5 CUT&RUN This paper GSE220570 FOXA2 ChIP-seq GEO GSE90456 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE90456 SOX2 ChIP-seq GEO GSE36570 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE36570 DNase-I hypersensitivity ENCODE Portal ENCFF737YIY https://www.encodeproject.org/files/ ENCFF737YIY/ Experimental models: Cell lines DH5A competent cells Invitrogen 18265017 Human BJ fibroblasts ATCC CRL-2522 (Continued on next page) 14 Cell Reports 42, 112748, July 25, 2023

Techniques: Diffusion-based Assay, Binding Assay

Journal: Cell reports

Article Title: Different chromatin-scanning modes lead to targeting of compacted chromatin by pioneer factors FOXA1 and SOX2.

doi: 10.1016/j.celrep.2023.112748

Figure Lengend Snippet: Figure 6. Non-DBD protein regions provide an essential contribution to the targeting and scanning of silent, compact chromatin by FOXA1 and SOX2 (A and B) SOX2- (A) and FOXA1-HALO/V5 (B) constructs and corresponding DBD truncations. (C and D) ChIP-seq and DNase-seq signals for SOX2, SOX2-DBD (C), and FOXA1, FOXA1-DBD (D), at shared peaks (top panel) or peaks bound only by the full-length proteins. (E) SMT measurement of scanning of low-mobility, compact chromatin by HALO-tagged SOX2 and FOXA1 full-length and DBD truncations. (F) Representative scanning trajectories by a single molecule of SOX2-DBD and FOXA1-DBD. (G and H) Average areas (mm2) after Delaunay triangulation of low-mobility, compact chromatin interactions spatial coordinates for 10,000 simu- lated scanning trajectories of full-length or DBD truncations of SOX2 (G) and FOXA1 (H). ***p < 0.0001, n.s., non-significant differences (p > 0.05) as determined by one-way ANOVA.

Article Snippet: Gel extraction kit QIAGEN 28706 Gibson Assembly kit NEB E5510 NEBNext UltraTM II for DNA Library Prep NEB E7645 CUTANATM pAG-MNase Epicypher 15–1016 Deposited data FOXA1-HALO ChIP-seq This paper GSE220570 HNF4A-HALO ChIP-seq This paper GSE220570 H2B-HALO 0h CHIP-seq This paper GSE220570 H2B-HALO 6h ChIP-seq This paper GSE220570 MNase-seq This paper GSE220570 SOX2-DBD-V5 CUT&RUN This paper GSE220570 FOXA1-NHAA-HALO ChIP-seq This paper GSE220570 FOXA1-RRAA-HALO ChIP-seq This paper GSE220570 FOXA1-DBD-HALO ChIP-seq This paper GSE220570 SOX2-V5 CUT&RUN This paper GSE220570 FOXA2 ChIP-seq GEO GSE90456 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE90456 SOX2 ChIP-seq GEO GSE36570 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE36570 DNase-I hypersensitivity ENCODE Portal ENCFF737YIY https://www.encodeproject.org/files/ ENCFF737YIY/ Experimental models: Cell lines DH5A competent cells Invitrogen 18265017 Human BJ fibroblasts ATCC CRL-2522 (Continued on next page) 14 Cell Reports 42, 112748, July 25, 2023

Techniques: Construct, ChIP-sequencing

Journal: Cancer Immunology Research

Article Title: Visualizing Spatial and Stoichiometric Barriers to Bispecific T-Cell Engager Efficacy

doi: 10.1158/2326-6066.cir-21-0594

Figure Lengend Snippet: Figure 1. Dose- and target-dependent huEGFRvIII/muCD3e-muFc BiTE function in vitro. A, General schematic depicting the surrogate BiTE design, consisting of an anti- huEGFRvIII TAA scFv domain, anti-murine CD3e scFv (murinized rat anti-mouse CD3e engager, KE3), and half-life extension moiety murine IgG1 single-chain Fc domain harboring a N297G mutation to minimize Fc effector function. MW, molecular weight; kDa: kilodaltons. B, Flow cytometry analysis of the coupling between mouse CD8þ T cells (violet proliferation dye (VPD)-labeled) and PyMT-Ctrl (control) or PyMT-EGFRvIII-Hi cells (CMTMR-labeled) cocultured for 1 hour in the presence of BiTEs as indicated. C, Calcium influx in mouse CD8þ T cells indicated by the ratio of Fura-2 signal, with comparison of excitation at 340 nm versus 380 nm. Left, representative images of T cells in contact with PyMT-EGFRvIII-Hi cells (close) and T cells that were further away (far) from cancer cells (scale bar, 10 mm). Right, quantification of the change of the Fura-2 ratio in the two T-cell populations (close and far), along with time. Also see Supplementary Video S1. N ¼ 10–20 cells per condition. D, Live imaging of active killing of PyMT-EGFRvIII-Hi tumor cells, labeled with a mix of live green dye and dead red dye, by VPD-labeled mouse CD8þ T cells in coculture for 2 hours in the presence of two concentrations of BiTEs (scale bar, 30 mm). Quantification of the number of dead cells over time is shown. Also see Supplementary Video S2. E, Flow cytometry analysis of mouse CD8þ T-cell proliferation (indicated by VPD dilution), activation (indicated by surface CD25 and CD69 expression), and tumor killing induced by BiTEs when T cells were cocultured with PyMT- EGFRvIII-Hi cells for 48 hours in the presence of different concentrations of BiTEs as indicated. Measurement of tumor cell death was done by staining with Live/Dead Zombie dye. N ¼ 3–4 per group. Data are representative of two independent experiments; , P < 0.0001; , P < 0.01; , P < 0.05 as determined by one-way ANOVA and multiple comparison test.

Article Snippet: To generate the PyMT-EGFRvIII cell line, a pLenti-humanEGFRvIII construct was generated by inserting the human EGFRvIII gene (NCBI Reference Sequence: NP_001333870.1) into the pLenti-puro vector (Addgene; #39481) and sequenced for accuracy.

Techniques: In Vitro, Mutagenesis, Molecular Weight, Flow Cytometry, Labeling, Control, Comparison, Imaging, Activation Assay, Expressing, Staining

Journal: Cancer Immunology Research

Article Title: Visualizing Spatial and Stoichiometric Barriers to Bispecific T-Cell Engager Efficacy

doi: 10.1158/2326-6066.cir-21-0594

Figure Lengend Snippet: Figure 2. BiTE-mediated target-binding and tumor killing in breast tumors. A, Schematic of the experimental design. PyMT-EGFRvIII-Hi cells and CD2-DsRed mice were used. Panels with corresponding data are indicated. B, Representative images showing BiTEs (20 mg) binding to T cells and tumor cells near vessels (< 10 mm). Blue outlines, tumor cells; red outlines, T cells. Scale bar, 15 mm. C, Quantification of individual T cell– or tumor cell–associated BiTE intensity over time. Each colored line represents one individual cell. Histogram (D) and quantification (E) of BiTE-AF488 binding on CD8þ T cells or PyMT-EGFRvIII-Hi mCherryþ tumor cells at two dosages and different time points as indicated. NT, no treatment. Representative flow plots (F) and quantification (G) of live tumor cells out of all CD45– live cells or all live cells in the tumors at 72 hours with high or low dosage of BiTEs. Live cells were gated using Zombie Aqua fixable viability dye and then mCherryþ cells were gated as live tumor cells. N ¼ 3 per group (D–G). Data are representative of two independent experiments; , P < 0.0001; , P < 0.001; , P < 0.01; , P < 0.05 as determined by one-way ANOVA and multiple comparison test.

Article Snippet: To generate the PyMT-EGFRvIII cell line, a pLenti-humanEGFRvIII construct was generated by inserting the human EGFRvIII gene (NCBI Reference Sequence: NP_001333870.1) into the pLenti-puro vector (Addgene; #39481) and sequenced for accuracy.

Techniques: Binding Assay, Comparison

Journal: Cancer Immunology Research

Article Title: Visualizing Spatial and Stoichiometric Barriers to Bispecific T-Cell Engager Efficacy

doi: 10.1158/2326-6066.cir-21-0594

Figure Lengend Snippet: Figure 3. Intratumoral vasculature diversity results in heterogeneous BiTE diffusion in solid tumors. A, Diagram showing protocol for intravital imaging of PyMT-EGFRvIII-Hi tumors. Representative images of AF488 fluorophore–labeled BiTE (BiTE-AF488, 20 mg injected at time ¼ 0) diffusion from vessels in a PyMT-EGFRvIII-Hi tumor. Three diffusion patterns are shown: Diffusive (B), irregular/biphasic (D), and contained (F). Scale bar, 50 mm. Also see Supplementary Video S4. C, E, and G, Corresponding quantifications of the BiTE accumulation at increasing distance from the vessels over time (color coded). H, The effective diffusion coefficient (Deff) for BiTE diffusion from vessels under conditions in panels B and D was calculated using the relationship R2 ¼ Deff t and plotted (see Materials and Methods). I, Plots of means of BiTE intensity on T cells over time at different distances away from vessels (color coded) in the diffusive and irregular diffusion patterns shown in B and D. Data are representative of two independent experiments.

Article Snippet: To generate the PyMT-EGFRvIII cell line, a pLenti-humanEGFRvIII construct was generated by inserting the human EGFRvIII gene (NCBI Reference Sequence: NP_001333870.1) into the pLenti-puro vector (Addgene; #39481) and sequenced for accuracy.

Techniques: Diffusion-based Assay, Imaging, Labeling, Injection

Journal: Cancer Immunology Research

Article Title: Visualizing Spatial and Stoichiometric Barriers to Bispecific T-Cell Engager Efficacy

doi: 10.1158/2326-6066.cir-21-0594

Figure Lengend Snippet: Figure 4. BiTE efficacy in solid tumors requires high target expression. A, Measurement of tumor size of indicated groups of mice over time. PyMT tumor cells [control (Ctrl), EGFRvIII-Hi, and EGFRvIII-Lo] were orthotopically injected into fat pad of the C57BL/6 mice. 10 mg BiTEs were injected intravenously (i.v.) at day 15 post tumor inoculation when tumors were palpable. B, Measurement of the tumor growth. Curves for individual mice shown for each group. C, Flow cytometry analysis of CD8þ

Article Snippet: To generate the PyMT-EGFRvIII cell line, a pLenti-humanEGFRvIII construct was generated by inserting the human EGFRvIII gene (NCBI Reference Sequence: NP_001333870.1) into the pLenti-puro vector (Addgene; #39481) and sequenced for accuracy.

Techniques: Expressing, Control, Injection, Flow Cytometry

Journal: Cancer Immunology Research

Article Title: Visualizing Spatial and Stoichiometric Barriers to Bispecific T-Cell Engager Efficacy

doi: 10.1158/2326-6066.cir-21-0594

Figure Lengend Snippet: Figure 6. Stimulatory DCs are required for BiTE efficacy in solid tumors. A, Measurement of PyMT-EGFRvIII-Hi tumor size for the indicated groups of mice over time. 10 mg BiTEs were injected intravenously (i.v.) at day 18 posttumor inoculation when tumors were palpable. B, Measurement of tumor growth from each individual mouse for the indicated groups. Tumors were harvested on day 24 posttumor injection. C, Flow cytometry analysis of DC populations. D, Quantification of IFNg and granzyme B secretion from tumoral CD8þ T cells for the indicated groups. Data are representative of two independent experiments; , P < 0.0001; , P < 0.001; , P < 0.01; , P < 0.05 as determined by the one-way ANOVA and multiple comparison test. NT, no treatment.

Article Snippet: To generate the PyMT-EGFRvIII cell line, a pLenti-humanEGFRvIII construct was generated by inserting the human EGFRvIII gene (NCBI Reference Sequence: NP_001333870.1) into the pLenti-puro vector (Addgene; #39481) and sequenced for accuracy.

Techniques: Injection, Flow Cytometry, Comparison