Fig. 1: SFN inhibits cell growth and promotes natural killer cell-mediated anti-tumor immune responses and killing of cHL cells through activation of the cGAS-STING pathway.

A Treatment of various cHL cell lines with increasing concentrations of SFN for 24 h resulted in decreased cell growth, shown as number of viable cells per mL of culture medium. Cell viability was also decreased at a concentration-dependent manner following SFN treatment for 24 h in cHL cells. The effects were variable among different cHL cell lines as shown. The cells were counted using trypan blue exclusion assay in triplicate. B Treatment with increasing concentrations of SFN for 24 h resulted in upregulation of IFN-β mRNA levels in all three cHL cell lines tested (L-1236, L-540, L-428) as assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using GAPDH as the housekeeping gene. The experiment was repeated at least two times. Statistical differences were assessed by two-paired t-test (** p < 0.01; **** p < 0.0001; ns, not significant). C Using a human cytokine array (105 different cytokines, chemokines, growth factors and other soluble proteins) performed in cell culture supernatants at 24 h post SFN treatment at a concentration of 40 μM, GM-CSF, RANTES and angiogenin (Supplementary Table 1) protein levels were consistently decreased in all three cHL cell lines tested (L-1236, L-540, L-428). By contrast, MIF was increased in L-540 and L-428 cells, but not in L-1236 cells (Supplementary Table 1). Differential effects on the cytokine profile were observed among various cHL cell lines. The relative mean spot pixel intensity was determined by image analysis with ImageJ. Statistical differences were assessed by two-paired t-test (Supplementary Table 1). D NK cell killing assessed with Chromium 51 (⁵¹Cr) release assay was significantly increased at 4–6 h of co-culture following treatment with 40 μM of SFN for 24 h in all cHL cell lines assessed. HUT78, a T-cell lymphoma cell line (Sézary syndrome), was included for comparison (Supplementary Fig. 3A). More specifically, percentage of specific killing at 9:1 effector-to-target (E:T) ratio is shown. Statistical differences were assessed by two-paired t-test (* p < 0.05; ** p < 0.01). E Profiling of NK cell ligand expression on HL cell lines treated with 40 uM of SFN for 24 h. A dramatic increase of MICA/B and a decrease of ULBP2/5/6 expression (NK ligands) was observed. Other ligands assessed did not show significant differences following SFN treatment. The relative mean fluorescence intensity (MFI) was determined by flow cytometry (FC) and presented as fold change compared to unstained samples. Statistical differences were assessed by two-paired t-test (** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant). F To confirm the functional significance of the MICA/B ligands in mediating the NK cell killing, the ⁵¹Cr release assay was performed after MICA/B gene silencing with or without additional SFN treatment at a concentration of 40 μM for 24 h in L-540 cells. RT-qPCR (left panel) using GAPDH as the housekeeping gene confirmed adequate silencing of the MICA gene in L-540 cells. Similar results were obtained for L-428 cell line (Supplementary Fig. 4). Statistical differences were assessed by two-paired t-test (* p < 0.05; **** p < 0.0001). G The schematic summarizes the effects of SFN on cell growth and anti-tumor immune responses in cHL. (left) SFN seems to inhibit cell cycle progression through upregulation of the CDK inhibitors p21 and p27, and downregulation of Cyclin D2. These effects might be mediated by activation (dephosphorylation) of SAMHD1 or SAMHD1-independent pathways. Similarly, SFN seems to reduce apoptosis through downregulation of BCL2, BCL-xL (inhibitors of endogenous or mitochondrial apoptotic pathway) as well as cFLIP (inhibitor of exogenous or death-receptor apoptotic pathway). (right) In addition, SFN promotes anti-tumor immune responses through expression of IFN-β, CXCL10 and the NK cell-activating ligands MICA/B. These effects maybe mediated by activation of cGAS-STING pathway-dependent or -independent mechanisms. SFN sulforaphane.