hg3 cells  (DSMZ)

Journal: Signal Transduction and Targeted Therapy

Article Title: Immunoglobulin heavy-chain status and stromal interactions shape ferroptosis sensitivity in chronic lymphocytic leukemia

doi: 10.1038/s41392-025-02535-x

Figure Lengend Snippet: a The CLL cell lines CII, HG3, I83-E95, JVM-3, Mec-1, PCL-12, PGA-1, and Wa-C3CD5+ were treated in two independent experiments with the GPX4 inhibitor ML162 (100 nM) or RSL3 (100 nM) for 4 h, and compound-triggered specific cell death was determined. Specific cell death was calculated relative to the untreated control as follows: 100 × (% dead cells − % baseline)/(100 − % baseline). The baseline values were normalized to 0%. b The same set of CLL cell lines was subjected to two independent experiments with the GPX4 inhibitors ML162 or RSL3 in the presence/absence of zVAD (apoptosis inhibitor, 10 µM), necrostatin-1/Nec-1 (necroptosis inhibitor, 100 µM), and deferoxamine (DFO, 100 µM) or ferrostatin-1/Fer-1 (ferroptosis inhibitors, 25 µM). c In the same samples, lipid peroxidation was assessed by flow cytometry (FACS) using BODIPY™ 581/591 C11, as shown in the representative analysis in the left panel. The data are summarized in the right panel. d Specific cell death of B cells from healthy donors (HD, n = 10) and CLL cells from patients (CLL, n = 20) was assessed via FACS following 24 h of ML162 treatment (500 nM), as shown in the representative analysis in the left panel. The data are summarized in the right panel. e Lipid peroxidation of B cells from healthy donors (HD, n = 10) and CLL cells from patients (CLL, n = 20) was assessed via FACS following 24 h of ML162 treatment (500 nM), as shown in the representative analysis in the left panel. The data are summarized in the right panel. f Expression of key pro- and anti-ferroptotic proteins was analyzed in primary CLL cells (CLL, n = 31) and HD B cells (HD, n = 12) on the basis of the median fluorescence intensity (MdFI) directly ex vivo. The data are shown as the fold change between CLL/HD patients. Statistical significance was determined on the basis of groupwise comparisons of MdFI values. Significantly differentially expressed proteins are highlighted in red. g Intracellular ferrous iron (Fe 2+ ) (HD, n = 10 and CLL, n = 24, left panel) and glutathione (HD, n = 8 and CLL, n = 34, right panel) baseline levels were measured via FACS via Phen Green SK and Thioltracker™, respectively. Note that the Phen Green SK signal is quenched by Fe²⁺; thus, lower fluorescence indicates higher intracellular ferrous iron levels. h Specific cell death was assessed in B cells and CLL cells from nontransgenic littermates (TCL1-, n = 5) and transgenic Eµ-TCL1 mice (TCL1+, n = 5) following ex vivo treatment for 24 h with 500 nM ML162. Statistical analysis: paired t-tests were applied for comparisons involving dependent (matched) samples ( a ), whereas unpaired t-tests were used for comparisons between independent groups ( d – h ). One-way ANOVA with multiple comparisons was used to assess differences across multiple treatment conditions ( b , c ). ‘n’ indicates the sample number, bars represent the mean; P-value: *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: HS-5 and HS-27A cells were obtained from ATCC (VA, USA), and CII, HG3, I83-E95, JVM-3, Mec-1, PCL-12, PGA-1, and Wa-C3CD5+ cells were obtained from DSMZ GmbH (Germany).

Techniques: Control, Flow Cytometry, Expressing, Fluorescence, Ex Vivo, Transgenic Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Immunoglobulin heavy-chain status and stromal interactions shape ferroptosis sensitivity in chronic lymphocytic leukemia

doi: 10.1038/s41392-025-02535-x

Figure Lengend Snippet: The CLL cell lines CII, HG3, I83-E95, and Mec-1 (three independent experiments) and primary CLL cells (orange squares, n = 10) were cultured in the presence/absence of HS-5 cells for 24 h and 48 h, respectively, followed by treatment with ML162 (100 nM), and a specific cell death and b lipid peroxidation were assessed by flow cytometry (FACS). Specific cell death was calculated relative to the untreated control as follows: 100 × (% dead cells − % baseline)/(100 − % baseline). The baseline values were normalized to 0%. c CLL cell lines CII, HG3, I83-E95, and Mec-1 (three independent experiments) and primary CLL cells (orange squares, n = 10) were cultured in the presence/absence of HS-5 cells for 24 h and 48 h, and the levels of intracellular thiols (i.e., glutathione) were assessed via FACS. Uptake of FITC-conjugated cystine (i.e., BioTracker TM cystine, CYS-BT) by CLL cells was evaluated under different conditions. First, d CII, HG3, I83-E95, Mec-1 (three independent experiments), and primary CLL cells (orange squares, n = 10) were cultured in the presence or absence of HS-5 cells for 24 h and 48 h, respectively. CYS-BT was added to the culture 30 min prior to measurement (=condition I). e CYS-BT was applied to the CLL cell lines following their coculture with and separation from HS-5 cells (=condition II). f CLL cell lines were cultured in the presence or absence of HS-5-derived CM, and CYS-BT was added to the medium (=condition III). g HS-5 cells were cultured in the presence of CYS-BT. The medium (including metabolized and secreted CYS-BT as well as any CYS-BT that was not taken up by HS-5 cells) was then collected and subsequently added to the culture of CLL cell lines (=condition IV). h Uptake of CYS-BT quantified on the basis of the CYS-BT median fluorescence intensity (MdFI) detected in CLL cells is summarized for all four conditions (cond.) I–IV. i CII, HG3, I83-E95, and Mec-1 (three independent experiments) were cocultured with/without HS-5 cells in the presence/absence of the inhibitor of GSH synthesis buthionine sulfoximine (BSO, 100 µM) for 24 h. Following treatment for 4 h with ML162 (100 nM), lipid peroxidation and specific cell death were assessed via FACS. j The left panel shows a representative FACS-based gating strategy for the CLL subpopulation of CD5 high CXCR4 low recent stromal emigrant (RSE) and CD5 low CXCR4 high long-term circulating (LTC) cells. Intracellular glutathione was semiquantified via ThiolTracker™ MdFI in RSE and LTC CLL cells from 30 patients, as shown in the right panel. Statistical analysis: Paired t-tests were applied for comparisons involving dependent (matched) samples ( a – g , j ), whereas one-way ANOVA with multiple comparisons was used to assess differences across multiple treatment conditions ( h , i ). ‘n’ indicates the sample number, bars represent the mean; P-value: *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: HS-5 and HS-27A cells were obtained from ATCC (VA, USA), and CII, HG3, I83-E95, JVM-3, Mec-1, PCL-12, PGA-1, and Wa-C3CD5+ cells were obtained from DSMZ GmbH (Germany).

Techniques: Cell Culture, Flow Cytometry, Control, Derivative Assay, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Immunoglobulin heavy-chain status and stromal interactions shape ferroptosis sensitivity in chronic lymphocytic leukemia

doi: 10.1038/s41392-025-02535-x

Figure Lengend Snippet: a The CLL cell lines CII, HG3, I83-E95, JVM-3, Mec-1, PCL-12, PGA-1, and Wa-C3CD5+ were cultured in two independent experiments for 24 h in the presence or absence of ibrutinib (Ibr, 10 µM) or venetoclax (Ven, 50 nM), with or without deferoxamine (DFO, 100 µM). Cell viability was assessed by flow cytometry (FACS), and specific types of cell death were determined. Specific cell death was calculated relative to that of the control (=baseline) as follows: 100 × (% dead cells − % baseline)/(100 − % baseline). The baseline values were normalized to 0%. b The contribution of ferroptosis to the overall cytotoxicity induced by ibrutinib or venetoclax is shown. The calculation is based on the rescue potential of the DFO. In addition, c we assessed the accompanying changes in lipid peroxidation and d relative changes in intracellular ferrous iron (Fe 2+ ) levels. e Primary CLL samples (n = 10, orange squares) were cultured for 24 h in the presence or absence of ibrutinib (Ibr, 10 µM) or venetoclax (Ven, 1 nM) with or without DFO (100 µM), and specific cell death and f the contribution of ferroptosis to overall cytotoxicity were assessed. g Patient-derived CLL samples (n = 10, orange squares) were cultured for 24 h in the presence or absence of ibrutinib (Ibr, 10 µM) or venetoclax (Ven, 1 nM) with or without zVAD (apoptosis inhibitor, 10 µM). Specific cell death was determined by FACS, and h the contribution of apoptosis to the overall cytotoxicity induced by ibrutinib or venetoclax is shown. The calculation is based on the rescue potential of zVAD. i CLL cell lines (red circles) pretreated for 24 h with/without ibrutinib (Ibr, 10 µM) or venetoclax (Ven, 50 nM) were subsequently treated with 100 nM ML162 or 100 nM RSL3, and specific cell death was assessed. j Primary CLL cells (orange squares) were pretreated for 24 h with/without ibrutinib (Ibr, 10 µM) or venetoclax (Ven, 1 nM) and subsequently treated with 500 nM ML162, and specific cell death (n = 18, left panel) and lipid peroxidation (n = 23, right panel) were assessed. k Relative gene expression of 88 ferroptosis-related genes was analyzed in the CLL cell lines CII, HG3, I83-E95, JVM-3, Mec-1, PCL-12, PGA-1, and Wa-C3CD5+, which were cultured for 24 h in the absence or presence of 10 µM ibrutinib, via a qPCR array. The volcano plot displays relative gene expression (log₂-fold change) versus −log₁₀ p-values, with red dots indicating genes whose expression was significantly upregulated (i.e., p-value ≤ 0.05 and at least 1.5-fold) upon ibrutinib treatment. l The CLL cell lines CII, HG3, I83-E95, JVM-3, Mec-1, PCL-12, PGA-1, and Wa-C3CD5+ were treated in two independent experiments for 24 h with or without 10 µM ibrutinib, and transferrin receptor (TFRC, CD71) surface protein was measured via FACS. m CLL cell lines CII, HG3, I83-E95, JVM-3, Mec-1, PCL-12, PGA-1, and Wa-C3CD5+ were treated in three independent experiments for 24 h with either an IgG isotype control (-) or an anti-CD71 blocking antibody (aCD71, 2 µg/mL) in the absence/presence of ibrutinib (Ibr, 10 µM), followed by GPX4 inhibition with 100 nM ML162 for 4 h. Specific cell death and n lipid peroxidation were assessed via FACS. o Splenocytes from transgenic Eµ-TCL1 mice (≥80% CLL cells) were adoptively transferred into wild-type recipients (n = 5 per group) via i.v. injection. Disease burden was monitored over an 8-week period by measuring CLL cell counts (cells/µL) via FACS. Treatments were initiated between weeks 3 and 4 (as indicated) and included ibrutinib (0.16 mg/mL in drinking water ad libitum), RSL3 (100 mg/kg i.p. twice weekly), or a combination of both. Statistical analysis: Paired t-tests were applied for comparisons involving dependent (matched) samples ( a , d , e , g , k , l ), whereas one-way ANOVA with multiple comparisons was used to assess differences across multiple treatment conditions ( c , i , j , m , n ). Two-way ANOVA was applied for analysis of experiments involving multiple factors, such as treatment conditions and time points ( o ). ‘n’ indicates the sample number, bars represent the mean, error bars represent the standard error of the mean; P-value: *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: HS-5 and HS-27A cells were obtained from ATCC (VA, USA), and CII, HG3, I83-E95, JVM-3, Mec-1, PCL-12, PGA-1, and Wa-C3CD5+ cells were obtained from DSMZ GmbH (Germany).

Techniques: Cell Culture, Flow Cytometry, Control, Derivative Assay, Gene Expression, Expressing, Blocking Assay, Inhibition, Transgenic Assay, Injection

Journal: Signal Transduction and Targeted Therapy

Article Title: Immunoglobulin heavy-chain status and stromal interactions shape ferroptosis sensitivity in chronic lymphocytic leukemia

doi: 10.1038/s41392-025-02535-x

Figure Lengend Snippet: a On the basis of a combination of their mutational status for TP53 (mutated, MT, and wild-type, WT) and IGHV (unmutated, U-CLL, and mutated, M-CLL), the CLL cell lines were grouped accordingly into the following: TP53 MT and U-CLL, TP53 WT and U-CLL, TP53 MT and M-CLL, and TP53 WT and M-CLL. Ferroptosis was triggered by 100 nM ML162 or 100 nM RSL3, and specific cell death was assessed. Specific cell death was calculated relative to that of the control (=baseline) as follows: 100 × (% dead cells − % baseline)/(100 − % baseline). The baseline values were normalized to 0%. b Lipid peroxidation levels were measured in U-CLL (CII, HG3, PCL-12, and Wa-C3CD5+) and M-CLL (I83-E95, JVM-3, MEC-1, and PGA-1) cells under basal conditions in three independent experiments. c U-CLL and M-CLL cell lines were treated with 100 nM ML162 or 100 nM RSL3 in two independent experiments, and lipid peroxidation was assessed. d Baseline Fe²⁺ content (left panel, three independent experiments) and its relative change upon treatment with 100 nM ML162 and 100 nM RSL3 (right panel, two independent experiments) were assessed in U-CLL and M-CLL cell lines via FACS via Phen Green SK. Note that the Phen Green SK signal is quenched by Fe²⁺; thus, lower fluorescence indicates higher intracellular ferrous iron levels. e Primary patient U-CLL (n = 10) and M-CLL (n = 10) cells were treated with 500 nM ML162, and specific cell death was assessed. f Baseline lipid peroxidation is shown for primary patient U-CLL (n = 12) and M-CLL cells (n = 12). g Baseline Fe²⁺ content was assessed in primary patient U-CLL (n = 13) and M-CLL cells (n = 12). h Primary patient U-CLL (n = 10-12) and M-CLL cells (n = 10-12) were pretreated for 24 h with ibrutinib (Ibr, 10 µM) and subsequently treated with 500 nM ML162, and specific cell death and lipid peroxidation were assessed. i The expression of key pro- and anti-ferroptotic proteins was compared in primary patient U-CLL (n = 11–17) and M-CLL cells (n = 12–17) on the basis of the median fluorescence intensity (MdFI). The data are shown as the fold change between M-CLL/U-CLL cells, and significantly altered proteins are highlighted in red. Statistical significance was determined on the basis of groupwise comparisons of MdFI values. j TFRC/CD71 protein levels in primary patient U-CLL and M-CLL cells were analyzed via data retrieved from the proteome dataset by Meier-Abt et al. k The left panel shows a representative FACS-based gating strategy for B-cell subsets in HD-derived peripheral blood (n = 10): naïve B cells (NaB), transitional B cells (TrB), non-switched memory B cells (NSM), and switched memory B cells (SM). The TFRC/CD71 levels of the corresponding B-cell subsets measured by FACS are summarized in the right panel. l Kaplan‒Meier analysis of treatment-free survival in CLL patients stratified by TFRC/CD71 expression levels, which was performed on the basis of publicly available proteome data from Meier-Abt et al. Statistical analysis: Unpaired t-tests were applied for comparisons between independent groups (Fig. 4b–j), whereas one‒way ANOVA with multiple comparisons was used to assess differences across multiple treatment conditions ( a , k ). Kaplan–Meier survival analysis was applied for survival comparisons ( l ). Abbreviations: ‘n’ indicates the sample number; bars represent the mean; P-value: *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: HS-5 and HS-27A cells were obtained from ATCC (VA, USA), and CII, HG3, I83-E95, JVM-3, Mec-1, PCL-12, PGA-1, and Wa-C3CD5+ cells were obtained from DSMZ GmbH (Germany).

Techniques: Control, Fluorescence, Expressing, Derivative Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Immunoglobulin heavy-chain status and stromal interactions shape ferroptosis sensitivity in chronic lymphocytic leukemia

doi: 10.1038/s41392-025-02535-x

Figure Lengend Snippet: a U-CLL (CII, HG3, PCL-12, and Wa-C3CD5+) and M-CLL (I83-E95, JVM-3, MEC-1, and PGA-1) cell lines were cultured for 24 h in three independent experiments in the absence (-) or presence (ART) of 10 µM artemisinin and then treated with 100 nM ML162, followed by an assessment of specific cell death and lipid peroxidation. Specific cell death was calculated relative to that of the control (=baseline) as follows: 100 × (% dead cells − % baseline)/(100 − % baseline). The baseline values were normalized to 0%. b U-CLL (HG3, CII) and M-CLL (Mec-1, I83-E95) cell lines were utilized in a drug screen with ferroptosis-related compounds at multiple dosages. For each compound, the median viability over all tested concentrations was calculated and expressed as a log₂-fold change comparing U-CLL to M-CLL cell lines, along with the corresponding −log₁₀ p-value. The volcano plot illustrates compounds that promote ferroptosis, with those exhibiting a significantly greater effect on U-CLL cell lines highlighted in red and those with a greater effect on M-CLL cell lines highlighted in green. c The dose‒response curve for linoleic acid is shown for the U-CLL (red) and M-CLL (green) cell lines (left panel). The corresponding area under the curve (AUC) is presented in the right panel. d ACSL1 and ACSL4 protein levels in primary patient U-CLL and M-CLL cells were analyzed via data retrieved from the proteome dataset by Meier-Abt et al. e U-CLL cell lines (HG3, CII, Wa-C3CD5+, and PCL-12) were cultured in two independent experiments for 24 h with or without linoleic acid (LA, 10 µM). Where indicated, Triacsin C (TrC, 5 µM) was added in combination with LA. Specific cell death was assessed by FACS. f The left panel shows a representative FACS-based viability analysis of U- and M-CLL cell lines that were cultured for 24 h in the absence or presence of linoleic acid (LA, 10 µM) and then treated with 100 nM ML162. The fold change in specific cell death in linoleic acid (LA)-pretreated cells compared with that in non-pretreated cells is summarized in the right panel for the U-CLL (HG3, CII) and M-CLL (Mec-1, I83-E95) cell lines. Each cell line was assessed in three independent experiments. g U-CLL cell lines (HG3, CII) were cultured in three independent experiments for 24 h in the absence or presence of linoleic acid (LA, 10 µM) and then treated with 100 nM ML162. Where indicated, Triacsin C (TrC, 5 µM) was added in combination with LA. The fold change in specific cell death in linoleic acid (LA)-pretreated cells compared with that in non-pretreated cells ± TrC is summarized in the panel. h Primary patient U-CLL cells (n = 8) were cultured for 24 h in the absence or presence of linoleic acid (LA, 10 µM) and then treated with 500 nM ML162. Specific cell death and lipid peroxidation were assessed by FACS. i U-CLL cell lines (CII, HG3, PCL-12, and Wa-C3CD5+) were treated in two independent experiments with linoleic acid (LA, 10 µM) and increasing dosages of the BTK inhibitor ibrutinib (0–25 µM). Specific cell death and lipid peroxidation were assessed by FACS. j Splenocytes from transgenic Eµ-TCL1 mice (≥80% CLL cells) were adoptively transferred into wild-type recipients (n = 5 per group) via i.v. injection. Disease burden was monitored over an 8-week period by measuring CLL cell counts (cells/µL) via FACS. Treatment with tung oil (100 µl/day p.o.) was initiated between weeks 3 and 4 (as indicated). Statistical analysis: Paired t-tests were applied for comparisons involving dependent (matched) samples ( a , e , g , and h ). Unpaired t-tests were applied for comparisons between independent groups ( b , d , and f ). Two-way ANOVA was applied for analysis of experiments involving multiple factors, such as treatment conditions and/or time points ( i , j ). ‘n’ indicates the sample number; bars represent the mean; error bars represent the standard error of the mean; P-value: *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: HS-5 and HS-27A cells were obtained from ATCC (VA, USA), and CII, HG3, I83-E95, JVM-3, Mec-1, PCL-12, PGA-1, and Wa-C3CD5+ cells were obtained from DSMZ GmbH (Germany).

Techniques: Cell Culture, Control, Transgenic Assay, Injection