human erythroleukemia cells  (ATCC)

Journal: Life Medicine

Article Title: Integration of a novel anti-PD-1 antibody with chimeric antigen receptor-T engineered to express interleukin-7 enhances targeting efficacy against lung cancer

doi: 10.1093/lifemedi/lnaf035

Figure Lengend Snippet: Engineering IL-7 in CAR-T cells enhances their anti-tumor activity .(A) Measurement of IL-7 secretion in culture supernatant by ELISA. Data are presented as the mean ± SD, n = 3, **** P < 0.0001. (B) Assessment of proliferation capacity of IL-7-CAR-T cells by cell count. NT, CAR-T, and IL-7-CAR-T cells were cultured with an initial cell count of 2 × 10 5 , and cell numbers were counted every other day. Data shown are the mean ± SD, n = 3; * P < 0.05, Student’s t -test. (C) The expression of CD45RA and CD62L on CAR-T cells and IL-7-CAR-T cells were analyzed by FACS. (D) Statistical analysis of memory T cell phenotypes. Data are shown as the mean ± SD, n = 3, * P < 0.05, Student’s t -test. (E) Evaluation of specific cytotoxicity of IL-7-CAR-T cells against lung cancer cell lines (HCC827, H23) using luciferase-based assays at various effector-to-target (E: T) ratios (10:1, 3:1, and 1: 1), with K562 cells as a negative control. Data are presented as the mean ± SD, n = 3, * P < 0.05, ** P < 0.01, Student’s t -test. (F) The experimental timeline for the treatment of cell line-derived xenograft (CDX) tumor model. 4 × 10 5 HCC827/Luc cells were subcutaneously injected into NPG mice. After 4 days, tumor-bearing NPG mice were treated intravenously with PBS, 1 × 10 7 NT, CAR-T, and IL-7-CAR-T cells. (G) Peripheral blood T-cell counts were quantified by flow cytometry at 10 days post-adoptive transfer. Data are shown as the mean ± SD, n = 3; ** P < 0.01, Student’s t -test. (H) Evaluation of the anti-tumor activity of IL-7-CAR-T cells by in vivo imaging in mice bearing HCC827 xenografts. Tumor growth was monitored over 28 days. (I) Bioluminescence signals from each mouse, as in (H), were recorded every 7 days. (J) The survival of tumor-bearing mice treated with PBS, NT, CAR-T cells and IL-7-CAR-T cells. PBS, CAR-T cells and IL-7-CAR-T cells group n = 5, NT group n = 3, * P < 0.05, ** P < 0.01, log-rank test. (K) Tumor-infiltrating T cell populations were quantified by flow cytometry at 10 days post-adoptive transfer. Data are shown as the mean ± SD, n = 3; **** P < 0.0001, Student’s t -test.

Article Snippet: The human lung cancer line NCI-H23 (H23, ATCC, CRL-5800) and human erythroleukemia cell line K562 (ATCC, CCL-243) were also obtained from ATCC and cultured in RPMI-1640 media with 10% fetal bovine serum under the same conditions.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Cell Counting, Cell Culture, Expressing, Luciferase, Negative Control, Derivative Assay, Injection, Flow Cytometry, Adoptive Transfer Assay, In Vivo Imaging

Journal: Life Medicine

Article Title: Integration of a novel anti-PD-1 antibody with chimeric antigen receptor-T engineered to express interleukin-7 enhances targeting efficacy against lung cancer

doi: 10.1093/lifemedi/lnaf035

Figure Lengend Snippet: Integration of IL-7-CAR-T with anti-PD-1 antibody significantly improves immunotherapy against lung cancer . (A) Detection of IL-2 and IFN-γ secretion in IL-7-CAR-T cells after co-culture with tumor cells (HCC827, H23, and K562) at an E : T ratio of 4:1, with 30 μg/mL C8A8. Iso and Nivo served as negative and positive controls, respectively. Data are shown as the mean ± SD, n = 3; *** P < 0.001, **** P < 0.0001, Student’s t -test. (B) C8A8 enhances the anti-tumor activity of IL-7-CAR-T cells in vitro . IL-7-CAR-T cells were co-incubated with HCC827, H23, and K562 cells at varying E: T ratios for 18 h with the presence of Iso, C8A8, and Nivo, respectively. Specific cytotoxicity was measured by Luciferase-based assays. Data are shown as the mean ± SD, n = 3; * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01, Student’s t -test. (C) Experimental timeline for the treatment of CDX tumor model using anti-PD-1 antibody and IL-7-CAR-T cells. NPG mice were injected subcutaneously with 4 × 10 5 HCC827/Luc cells. After 4 days, mice bearing tumors were intravenously infused with 5 × 10 6 NT and IL-7-CAR-T cells, respectively. The mice were intravenously injected with C8A8 (5 mg/kg, n = 5) or Nivo (5 mg/kg, n = 5) or isotype control antibody (5 mg/kg, n = 5) on day 0, 3, 6, and 9. (D) Representative images of tumor regression in tumor-bearing mice treated. Monitoring of tumor growth over 28 days. (E) Bioluminescence signals of each mouse from (D) were recorded at the indicated time points. (F) Combination therapy of IL-7-CAR-T cells with anti-PD-1 antibodies significantly prolongs the survival of tumor-bearing mice compared to the control groups. Data are shown as the mean ± SD; * P < 0.05, log-rank test.

Article Snippet: The human lung cancer line NCI-H23 (H23, ATCC, CRL-5800) and human erythroleukemia cell line K562 (ATCC, CCL-243) were also obtained from ATCC and cultured in RPMI-1640 media with 10% fetal bovine serum under the same conditions.

Techniques: Co-Culture Assay, Activity Assay, In Vitro, Incubation, Luciferase, Injection, Control

Journal: Cell Reports Medicine

Article Title: Multiplex base editing of BCL11A regulatory elements to treat sickle cell disease

doi: 10.1016/j.xcrm.2025.102376

Figure Lengend Snippet: Evaluation of on-target aberrations and off-target activity of multiplex base editing of +58-kb and +55-kb regions (A and B) C-G to T-A or/and A-T to G-C base-editing efficiency in the +58-kb (A) and +55-kb (B) target region obtained with the generation of +58 CBEI/+55 CBEII profile and +58 DBEI/+55 DBEII. (C) Comprehensive allele frequencies of diverse types of genomic rearrangements and InDel types in SCD HSPC-derived erythroblasts. We reported frequencies of events observed at individual GATA1 and ATF4 BSs (in the absence of the 3.2-kb deletion/inversion) and the frequencies of 3.2-kb deletion/inversion (GATA1-ATF4). InDels were classified as small, intermediate, and large according to their size ([4–50 bp], [51–200 bp], and >200 bp). Del, deletion; Ins, insertion; Inv, inversion. Unmodified, no deletion, inversion, or insertion observed. (D) Frequency of alleles edited at the GATA1 and ATF4 BSs, either simultaneously (GATA1 and ATF4) or at only one of the sites (GATA1 or ATF4). The reported data were adjusted for background error typical of this analysis by subtracting the frequency of edited alleles in the mock condition from the fraction of edited alleles in the edited conditions. (E) sgRNA-dependent off-target sites of the GATA_bs_1 (top) and ATF4_bs_2 (bottom) sgRNAs in K562 cells, as evaluated by GUIDE-seq analysis. The protospacer targeted by each sgRNA is reported on top of each panel, followed by the off-target sites and their mismatches with the on-target (highlighted in color). The number of sequencing reads (abundance), the number of mismatches, and the chromosomal coordinates (hg38) of each off-target site are reported. (F and G). Base editing and InDel frequency at on-target and off-target (OT) sites, in SCD HSPC-derived erythroblasts, were evaluated for GATA1 BS targeting sgRNA (F) and ATF4 BS targeting sgRNA (G) for control, +58 CBEI/+ 55 CBEII and +58 DBEI/+55 DBEI profiles as measured by targeted NGS. OT sites in introns or intergenic regions for which we detected editing are highlighted by a red or blue square shape, respectively. Data are expressed as individual values and median ( n = 3 biologically independent experiments, 3 donors) ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. Two-way ANOVA with Dunnett’s statistical significance between mock and edited samples is depicted in the graph. (H and I) Strip plots showing the variant allele frequency (A > G, H; C > T, I) in exons, observed in SCD HSPCs obtained from three different donors and measured by WES. The total number of variants are indicated above each sample. (J) Volcano plots showing differential gene expression between cells treated with CBE, DBE, or Cas9 nuclease and mock-treated samples. The horizontal dashed line indicates the threshold on the false discovery rate (FDR ≤ 0.05), and the vertical dashed lines correspond to the threshold on log2FC ≥ 1 or ≤ −1. Upregulated genes are indicated in red, and downregulated genes are in blue. Genes in gray are not differentially expressed. ( n = 1 biologically independent experiments, 3 donors). (K and L) Strip plots showing the variant allele frequency (A > G, K; C > T, L) in the transcriptome observed in SCD HSPCs obtained from three different donors and measured by RNA-seq. The total number of variants are indicated above each sample.

Article Snippet: K562 human erythroleukemia cell line , ATCC , Cat #CCL-243.

Techniques: Activity Assay, Multiplex Assay, Derivative Assay, Sequencing, Control, Stripping Membranes, Variant Assay, Gene Expression, RNA Sequencing

Journal: Cell Reports Medicine

Article Title: Multiplex base editing of BCL11A regulatory elements to treat sickle cell disease

doi: 10.1016/j.xcrm.2025.102376

Figure Lengend Snippet: Evaluation of on-target aberrations and off-target activity of multiplex base editing of +58-kb and +55-kb regions (A and B) C-G to T-A or/and A-T to G-C base-editing efficiency in the +58-kb (A) and +55-kb (B) target region obtained with the generation of +58 CBEI/+55 CBEII profile and +58 DBEI/+55 DBEII. (C) Comprehensive allele frequencies of diverse types of genomic rearrangements and InDel types in SCD HSPC-derived erythroblasts. We reported frequencies of events observed at individual GATA1 and ATF4 BSs (in the absence of the 3.2-kb deletion/inversion) and the frequencies of 3.2-kb deletion/inversion (GATA1-ATF4). InDels were classified as small, intermediate, and large according to their size ([4–50 bp], [51–200 bp], and >200 bp). Del, deletion; Ins, insertion; Inv, inversion. Unmodified, no deletion, inversion, or insertion observed. (D) Frequency of alleles edited at the GATA1 and ATF4 BSs, either simultaneously (GATA1 and ATF4) or at only one of the sites (GATA1 or ATF4). The reported data were adjusted for background error typical of this analysis by subtracting the frequency of edited alleles in the mock condition from the fraction of edited alleles in the edited conditions. (E) sgRNA-dependent off-target sites of the GATA_bs_1 (top) and ATF4_bs_2 (bottom) sgRNAs in K562 cells, as evaluated by GUIDE-seq analysis. The protospacer targeted by each sgRNA is reported on top of each panel, followed by the off-target sites and their mismatches with the on-target (highlighted in color). The number of sequencing reads (abundance), the number of mismatches, and the chromosomal coordinates (hg38) of each off-target site are reported. (F and G). Base editing and InDel frequency at on-target and off-target (OT) sites, in SCD HSPC-derived erythroblasts, were evaluated for GATA1 BS targeting sgRNA (F) and ATF4 BS targeting sgRNA (G) for control, +58 CBEI/+ 55 CBEII and +58 DBEI/+55 DBEI profiles as measured by targeted NGS. OT sites in introns or intergenic regions for which we detected editing are highlighted by a red or blue square shape, respectively. Data are expressed as individual values and median ( n = 3 biologically independent experiments, 3 donors) ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. Two-way ANOVA with Dunnett’s statistical significance between mock and edited samples is depicted in the graph. (H and I) Strip plots showing the variant allele frequency (A > G, H; C > T, I) in exons, observed in SCD HSPCs obtained from three different donors and measured by WES. The total number of variants are indicated above each sample. (J) Volcano plots showing differential gene expression between cells treated with CBE, DBE, or Cas9 nuclease and mock-treated samples. The horizontal dashed line indicates the threshold on the false discovery rate (FDR ≤ 0.05), and the vertical dashed lines correspond to the threshold on log2FC ≥ 1 or ≤ −1. Upregulated genes are indicated in red, and downregulated genes are in blue. Genes in gray are not differentially expressed. ( n = 1 biologically independent experiments, 3 donors). (K and L) Strip plots showing the variant allele frequency (A > G, K; C > T, L) in the transcriptome observed in SCD HSPCs obtained from three different donors and measured by RNA-seq. The total number of variants are indicated above each sample.

Article Snippet: Human erythroleukemia K562 cells were obtained from ATCC (CCL-243), authenticated by the vendor, and tested for mycoplasma contamination.

Techniques: Activity Assay, Multiplex Assay, Derivative Assay, Sequencing, Control, Stripping Membranes, Variant Assay, Gene Expression, RNA Sequencing