Fig. 2: Stromal cells increase CLL cell resilience to ferroptosis.

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^highCXCR4^low recent stromal emigrant (RSE) and CD5^lowCXCR4^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