Journal: Cell Reports Medicine

Article Title: Sonogenetics-controlled synthetic designer cells for cancer therapy in tumor mouse models

doi: 10.1016/j.xcrm.2024.101513

Figure Lengend Snippet: Cytotoxic effects of azurin secreted by US-mediated VNP20009 cells against melanoma and colorectal cancer cells (A) Schematic representation of SINGER-mediated protein expression and secretion in VNP20009 cells. (B) The expression and secretion of a Gaussia luciferase (Gluc) reporter in VNP20009 cells after US stimulation. (C) Schematic for the time schedule and experimental procedure for assessing the reversibility of the SINGER system. (D) Reversibility of SINGER-mediated transgene expression. VNP20009 cells (OD 600 = 0.25) transformed with pGT240 were cultivated for 36 h while alternating irradiation with US (0.5 W/cm 2 , pulse of 1 s on, 1 s off) for 1 h or without US. Gluc expression in the culture supernatants was quantified every 2 h. Cell density was adjusted to OD 600 = 0.25 when the pattern changed. (E) Schematic of the experimental procedure for analysis of secretion proteins. (F and G) The expression and secretion of azurin in VNP20009 cells after US stimulation. Azurin in the culture supernatants was checked by immunoblotting analysis (F) and ELISA (G). (H) Measurement of cell viability by CCK-8 assays. B16F10 and CT26 cells were treated with the culture supernatants of VNP20009 harboring SINGER-azurin with (right) or without (left) US stimulation. (I) Fluorescence microscopy images for the detection of apoptosis by TUNEL assay. Scale bar, 50 μm. (J–L) Representative flow cytometry plots using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining for apoptosis (J and K) and quantification of apoptosis (L). Data in (B), (D), (G), (H), and (L) are presented as means ± SD; n = 3 independent experiments. Each data point represents the mean of three technical replicates. p values were calculated by one-way analysis of variance (ANOVA) with Tukey’s post-test. ∗∗∗ p < 0.001. See also Figures S2–S4 .

Article Snippet: B16F10 luci (Luciferase tagged mouse B16F10 melanoma cell line), B16F10 melanoma cell line (Meisen CTCC) and mouse colon carcinoma cell line (CRL-2638, ATCC) were cultured in RPMI-1640 medium (11875093, Gibco) with 10% (v/v) fetal bovine serum (FBS; 16000-044, Gibco) and 1% (v/v) penicillin/streptomycin (ST488-1/ST488-2, Beyotime).

Techniques: Expressing, Luciferase, Transformation Assay, Irradiation, Western Blot, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Fluorescence, Microscopy, TUNEL Assay, Flow Cytometry, Staining

Journal: Cell Reports Medicine

Article Title: Sonogenetics-controlled synthetic designer cells for cancer therapy in tumor mouse models

doi: 10.1016/j.xcrm.2024.101513

Figure Lengend Snippet: SINGER-mediated transgene expression in a tumor xenograft mouse model (A) Schematic illustration of the experimental design for evaluating SINGER-mediated transgene expression in tumors. (B and C) Time-course-dependent SINGER-mediated transgene expression kinetics in tumors revealed by bioluminescence images in mice. C57BL/6 mice bearing B16F10 melanoma tumors received an intratumoral injection of VNP20009 cells (5 × 10 5 colony-forming units [CFUs]) transformed with pGT240, after which the mice were exposed to US (0.5 W/cm 2 , pulse with 1 s on, 1 s off) for the indicated durations. The bioluminescence signal intensity was quantified with an in vivo imaging system 12 h after US irradiation. The data in (C) are presented as means ± SEM; n = 4 mice. p values were calculated by one-way ANOVA with Tukey’s post-test. ∗∗ p < 0.01 and ∗∗∗ p < 0.001. (D) Immunoblotting for azurin levels in tumor tissues. (E) Immunofluorescence staining of tumor sections. The azurin expressed and secreted by US-mediated VNP20009 cells was stained with an anti-FLAG antibody (red). Bacterial cells were stained with an anti-DnaK antibody (green). Nuclei were stained with DAPI (blue). Azurin-FLAG and DnaK were detected by fluorescence confocal microscopy. Scale bar, 10 μm. See also Figures S5–S7 .

Article Snippet: B16F10 luci (Luciferase tagged mouse B16F10 melanoma cell line), B16F10 melanoma cell line (Meisen CTCC) and mouse colon carcinoma cell line (CRL-2638, ATCC) were cultured in RPMI-1640 medium (11875093, Gibco) with 10% (v/v) fetal bovine serum (FBS; 16000-044, Gibco) and 1% (v/v) penicillin/streptomycin (ST488-1/ST488-2, Beyotime).

Techniques: Expressing, Injection, Transformation Assay, In Vivo Imaging, Irradiation, Western Blot, Immunofluorescence, Staining, Fluorescence, Confocal Microscopy

Journal: Cell Reports Medicine

Article Title: Sonogenetics-controlled synthetic designer cells for cancer therapy in tumor mouse models

doi: 10.1016/j.xcrm.2024.101513

Figure Lengend Snippet: Antitumor performance of VNP20009 cells harboring SINGER-azurin in mice bearing B16F10 luci melanoma tumors (A) Schematic illustrating the experimental design for evaluating the antitumor performance of VNP20009 cells harboring SINGER-azurin against B16F10 luci melanoma tumors. C57BL/6 mice bearing B16F10 luci melanoma tumors received intratumoral injection of PBS alone, US alone (US), untransformed VNP20009 cells (VNP20009), untransformed VNP20009 cells with US (VNP20009+US), VNP20009 cells harboring SINGER-azurin with US induction (SINGER-azurin+US), or VNP20009 cells harboring SINGER-azurin without US induction (SINGER-azurin). (B) Serial in vivo bioluminescence imaging of B16F10 luci -tumor-bearing mice in the groups depicted in (A). Five representative mice per treatment group are shown. (C) Bioluminescence quantification of B16F10 luci -tumor-bearing mice treated with different treatments. (D) Tumor volume measurement in the indicated mouse groups. (E) Kaplan-Meier curves for mouse survival. (F) Individual growth curves of B16F10 luci melanoma tumors in different mouse groups. Data in (C)–(E) are presented as means ± SEM; n = 5 mice per group. p values were calculated by one-way ANOVA with Tukey’s post-test (∗∗∗∗ p < 0.0001).

Article Snippet: B16F10 luci (Luciferase tagged mouse B16F10 melanoma cell line), B16F10 melanoma cell line (Meisen CTCC) and mouse colon carcinoma cell line (CRL-2638, ATCC) were cultured in RPMI-1640 medium (11875093, Gibco) with 10% (v/v) fetal bovine serum (FBS; 16000-044, Gibco) and 1% (v/v) penicillin/streptomycin (ST488-1/ST488-2, Beyotime).

Techniques: Injection, In Vivo, Imaging

Journal: Cell Reports Medicine

Article Title: Sonogenetics-controlled synthetic designer cells for cancer therapy in tumor mouse models

doi: 10.1016/j.xcrm.2024.101513

Figure Lengend Snippet: SINGER-mediated antitumor performance of engineered VNP20009 delivered via intravenous injection in different tumor xenograft mouse models (A) Schematic illustration of SINGER-mediated antitumor performance against B16F10 luci melanoma tumors. C57BL/6 mice bearing B16F10 luci melanoma tumors received intravenous injections of PBS, VNP20009, US, VNP20009+US, SINGER-azurin, or SINGER-azurin+US. (B) Serial in vivo bioluminescence imaging of B16F10 luci tumors expressing luciferase in the groups described in (A). (C) Bioluminescence quantification of B16F10 luci -tumor-bearing mice treated with different treatments. (D) Tumor volume measurement in the indicated mouse groups. (E) Kaplan-Meier curves for mouse survival in (B). (F–H) Tumor volume measurement in mice bearing various xenograft tumors, including CT26 (F), A20 (G), and H22 tumors (H). (I–K) Kaplan-Meier curves for mouse survival in (F)–(H). Data in (C)–(K) are presented as means ± SEM; n = 4–6 mice. p values were calculated by one-way ANOVA with Tukey’s post-test (∗∗∗∗ p < 0.0001). See also Figures S8–S11 .

Article Snippet: B16F10 luci (Luciferase tagged mouse B16F10 melanoma cell line), B16F10 melanoma cell line (Meisen CTCC) and mouse colon carcinoma cell line (CRL-2638, ATCC) were cultured in RPMI-1640 medium (11875093, Gibco) with 10% (v/v) fetal bovine serum (FBS; 16000-044, Gibco) and 1% (v/v) penicillin/streptomycin (ST488-1/ST488-2, Beyotime).

Techniques: Injection, In Vivo, Imaging, Expressing, Luciferase

Journal: Cell Reports Medicine

Article Title: Sonogenetics-controlled synthetic designer cells for cancer therapy in tumor mouse models

doi: 10.1016/j.xcrm.2024.101513

Figure Lengend Snippet:

Article Snippet: B16F10 luci (Luciferase tagged mouse B16F10 melanoma cell line), B16F10 melanoma cell line (Meisen CTCC) and mouse colon carcinoma cell line (CRL-2638, ATCC) were cultured in RPMI-1640 medium (11875093, Gibco) with 10% (v/v) fetal bovine serum (FBS; 16000-044, Gibco) and 1% (v/v) penicillin/streptomycin (ST488-1/ST488-2, Beyotime).

Techniques: Virus, Recombinant, Lysis, Sterility, Plasmid Preparation, Modification, Red Blood Cell Lysis, Staining, Enzyme-linked Immunosorbent Assay, Cloning, CCK-8 Assay, Double Staining, Bicinchoninic Acid Protein Assay, TUNEL Assay, Apoptosis Assay, Generated, Software, Western Blot, Imaging

Journal: Cell Reports

Article Title: Restoration of visual function in advanced disease after transplantation of purified human pluripotent stem cell-derived cone photoreceptors

doi: 10.1016/j.celrep.2021.109022

Figure Lengend Snippet:

Article Snippet: Plasmid: L-MYC and LIN28 episonal insert (pCXLE-hUL) , , Addgene plasmid #27080.

Techniques: Virus, Recombinant, Plasmid Preparation, Software

Journal: Nucleic Acids Research

Article Title: Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation

doi: 10.1093/nar/gkad562

Figure Lengend Snippet: TSS-MPRA of synthetic regulatory sequences mirrors the vast majority of endogenous initiation patterns and transcription levels. ( A ) Schematic of TSS-MPRA. Transcription activity and location of transcription initiation is determined by 5′ RNA-seq of reporter transcripts initiating within synthetic DNA inserts cloned into reporter plasmids and electroporated into cells. Histograms on the right show cumulative DNA-normalized number of sequence tags aligning to each base position of a 153-bp region surrounding the human HBE1 promoter in K562 cells. Capped reporter transcripts are drawn in purple. RT: reverse transcription primer landing site. BC: barcode sequence. ( B ) Spearman's correlation of DNA-normalized RNA levels of all inserts of two replicate episomal TSS-MPRA experiments. ( C ) Correlation between the transcriptional signal of 250 genomic DNA inserts in epi-short TSS-MPRA and csRNA-seq of the corresponding endogenous loci. TSS-MPRA inserts were chosen randomly from locations exhibiting transcription activity as measured by csRNA-seq. Spearman's correlation of TSS-MPRA and csRNA-seq transcription levels of all regulatory sequences, or of promoters (red) or enhancers (blue, outside of a ± 2-kb window of RefSeq-annotated promoters). Regions were chosen to cover a wide range of transcription levels and initiation patterns. Each dot represents the relative transcript levels observed in each assay as total normalized read counts of all transcripts aligning to each region. TSS-MPRA RNA read counts were normalized by the corresponding plasmid DNA read counts.

Article Snippet: The episomal TSS-MPRA plasmid is based on the background-reduced pGL4.10 luciferase reporter plasmid (Promega).

Techniques: Activity Assay, RNA Sequencing Assay, Clone Assay, Sequencing, Reverse Transcription, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation

doi: 10.1093/nar/gkad562

Figure Lengend Snippet: TSS-MPRA fidelity correlates with genomic TSS pattern width, transcription level, and presence of core promoter elements. ( A ) Schematic of the outlier detection model used to determine whether two TSS distributions are similar or not. Higher WIP scores (see methods for derivation) are indicative of more dissimilar initiation patterns. ( B ) TSS-MPRA preferentially recapitulates focused initiation patterns. Focus ratios (y-axis) of TSS-MPRA initiation patterns that are similar (blue) or dissimilar (red) to the corresponding endogenous initiation patterns as measured by csRNA-seq. Focus ratios of 0 indicate fully dispersed (broad) initiation patterns, while 1 indicates fully focused (sharp) patterns. ( C ) TSS-MPRA better recapitulates initiation patterns of more actively transcribed genomic regions. Endogenous locus transcription levels (csRNA-seq tag counts, y-axis) where TSS-MPRA initiation shape is similar (blue) or dissimilar (red) to the corresponding endogenous initiation pattern. ( D ) Strong transcription in the TSS-MPRA correlates with presence of TATA and Inr core promoter elements. Position-specific nucleotide frequencies (y-axis) (A: blue, C: purple, G: red, and T: orange) relative to each TSS in all TSS-MPRA inserts where: overall TSS-MPRA shapes of the inserts mirror the endogenous initiation patterns (I), or where insert TSS shapes do not mirror endogenous initiation patterns and either the respective TSSs within the overall TSS shape have 3x higher contribution to the overall signal of a given insert in TSS-MPRA data than in csRNA-seq (II), or the respective TSSs have 3× higher contribution to the overall signal of a given insert in csRNA-seq over TSS-MPRA data (III). The x-axis denotes the distance in bp from each TSS (bp 0).

Article Snippet: The episomal TSS-MPRA plasmid is based on the background-reduced pGL4.10 luciferase reporter plasmid (Promega).

Techniques:

Journal: Nucleic Acids Research

Article Title: Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation

doi: 10.1093/nar/gkad562

Figure Lengend Snippet: Longer inserts initiate transcription at additional non-endogenous TSSs and decrease overall TSS-MPRA transcription initiation fidelity. ( A ) Non-native TSS use in epi-long TSS-MPRA. Frequency of TSS usage between csRNA-seq (top), short TSS-MPRA (middle), and long TSS-MPRA (bottom). The y-axis, TSS usage frequency, is defined as the oligo position-specific cumulative normalized initiation frequencies in TSS-MPRA and csRNA-seq across all native TSS-MPRA inserts and corresponding genomic regions. At the top a schematic representation of the insert-containing oligos: overhang cloning sequence, followed by genomic DNA insert, 11-mer barcode and second overhang cloning sequence. The box and dots in blue marked ‘CORE’ are the positions covered by the 153-bp insert of the epi-short pool. ( B ) Increased insert length increases enhancer but not promoter transcription correlation between TSS-MPRA and csRNA-seq. Spearman's correlation of epi-long TSS-MPRA and csRNA-seq levels between all (purple) 250 regulatory sequences selected to cover a wide range of transcription levels and initiation patterns, or of only enhancers (blue), or only promoters (red). ( C ) Increased correlation of CORE (153-bp region) initiation frequencies within longer inserts of enhancers but not promoters. This analysis is restricted to the 153-bp region marked ‘CORE’ in (A). Color scheme as in (B).

Article Snippet: The episomal TSS-MPRA plasmid is based on the background-reduced pGL4.10 luciferase reporter plasmid (Promega).

Techniques: Clone Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation

doi: 10.1093/nar/gkad562

Figure Lengend Snippet: Reporter chromatinization has negligible effect on transcription initiation patterns and lowers correlation between TSS-MPRA and csRNA-seq transcription levels. ( A ) Schematic of genomic integration of a synthetic insert using lentiviral integration. ( B ) High reproducibility of Lenti-TSS-MPRA. Spearman's correlation of RNA/DNA normalized levels between all the inserts of two replicate lentiviral TSS-MPRA experiments. ( C ) High reproducibility in TSS profile changes between episomal and lentiviral TSS-MPRA experiments. WIP scores between csRNA-seq and lentiviral TSS-MPRA on the y-axis are highly correlated with the WIP scores between csRNA-seq and episomal TSS-MPRA on the x-axis. Dots are colored by how dissimilar insert TSS profiles are between lentiviral and episomal TSS-MPRA experiments. Higher WIP scores represent greater dissimilarity. ( D ) Spearman's correlation of lenti-short Lenti-TSS-MPRA and csRNA-seq levels between all 250 randomly selected regulatory sequences covering a wide range of transcription levels and initiation patterns (purple), only enhancers (blue), or only promoters (red). ( E ) Spearman's correlation of lenti-long Lenti-TSS-MPRA and csRNA-seq levels between all 250 randomly selected regulatory sequences covering a wide range of transcription levels and initiation patterns (purple), only enhancers (blue) or only promoters (red). ( F ) Spearman's correlation of lenti-long Lenti-TSS-MPRA and csRNA-seq levels between all 250 randomly selected regulatory sequences covering a wide range of transcription levels and initiation patterns (purple), only enhancers (blue), or only promoters (red). This analysis is restricted to the 153-bp region marked ‘CORE’ in Figure .

Article Snippet: The episomal TSS-MPRA plasmid is based on the background-reduced pGL4.10 luciferase reporter plasmid (Promega).

Techniques:

Journal: Nucleic Acids Research

Article Title: Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation

doi: 10.1093/nar/gkad562

Figure Lengend Snippet: TSS-MPRA enables studying the effect of motif mutations on reporter-driven initiation patterns and transcription levels. ( A ) Tracking transcription initiation changes caused by mutations in transcription factor and core promoter element motifs in episomal plasmids. Scatterplot comparing the changes in initiation patterns (WIP score, y-axis) and transcription levels (fold change, x-axis) between control and mutated inserts in episomal plasmids. Red dots signify inserts with significantly changed TSS shapes after mutation. ( B ) TSS shape changes after motif mutation in episomal constructs. The y-axis represents the mean WIP score between all inserts (and their barcode replicates) containing a particular motif and the corresponding insert with the mutated motif. Colors correspond to motif identities. ( C ) Transcription level changes associated with motif mutation in episomal constructs. The y-axis represents the mean fold transcription change of the inserts (and their barcode replicates) containing a particular wild-type or mutated motif (x-axis). ( D ) Tracking transcription initiation changes caused by transcription factor and core promoter element motif mutations after lentiviral integration into the genome. Scatterplot comparing the changes in initiation patterns (WIP score, y-axis) and transcription levels (fold change, x-axis) between control and mutated inserts in lentiviral plasmids. Red dots signify inserts with significantly changed TSS shapes after mutation. ( E ) TSS shape changes after motif mutation in lentiviral constructs. The y-axis represents the mean WIP score between all inserts (and their barcode replicates) containing a particular motif and the corresponding insert with the mutated motif. Colors correspond to motif identities. ( F ) Transcription level changes associated with motif mutation in lentiviral constructs. The y-axis represents the mean fold transcription change of the inserts (and their barcode replicates) containing a particular wild-type or mutated motif (x-axis). ( G ) Example track for the ACTB promoter, showing csRNA-seq (top), TSS-MPRA, and Lenti-TSS-MPRA output without (black) and with (red) TATA-box motif mutation. Blue highlights indicate the positions where motifs were replaced by a constant sequence with no known transcription factor motif. ( H ) Example track for the ENO1 promoter, showing csRNA-seq (top), TSS-MPRA, and Lenti-TSS-MPRA output before (black) and after (red) PU.1 motif mutation. Blue highlights indicate the positions where motifs were replaced.

Article Snippet: The episomal TSS-MPRA plasmid is based on the background-reduced pGL4.10 luciferase reporter plasmid (Promega).

Techniques: Control, Mutagenesis, Construct, Sequencing

Journal: Nucleic Acids Research

Article Title: Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation

doi: 10.1093/nar/gkad562

Figure Lengend Snippet: Assessing the effects of single nucleotide polymorphisms on reporter-driven initiation patterns and transcription levels. ( A ) Allele-specific transcription initiation differences caused by known GWAS SNPs in episomal plasmids. Scatterplot comparing the changes in initiation patterns (y-axis) and transcription fold change (x-axis) between control and variant inserts in episomal constructs. Red dots signify inserts that had significant changes to their TSS shapes. ( B ) Allele-specific transcription initiation differences caused by known GWAS SNPs in lentiviral constructs. Scatterplot comparing the changes in initiation patterns (y-axis) and transcription fold change (x-axis) between control and variant inserts in lentivirally integrated constructs. Red dots signify inserts that had significant changes to their TSS shapes. ( C ) SNP rs1991401 is associated with TSS shape changes. Track showing csRNA-seq (top), TSS-MPRA, and Lenti-TSS-MPRA output of the T (black) and G (red) allele. A blue ‘^’ symbol indicates the location of the SNP. ( D ) TSS shape differences associated with SNP rs131804. Track showing csRNA-seq (top), TSS-MPRA, and Lenti-TSS-MPRA output of the C (black) and A (red) allele. A blue ‘^’ symbol marks the SNP location.

Article Snippet: The episomal TSS-MPRA plasmid is based on the background-reduced pGL4.10 luciferase reporter plasmid (Promega).

Techniques: Control, Variant Assay, Construct