Extended Data Fig. 6: 3D-printed HCC model demonstrated the role of SPON2 in promoting the activation and migration of NK92 cells.

a, b, Representative images depicting NK92 cell migration toward the tumor region. NK92 cells were fluorescently labelled with PKH26 (in red), and HepG2 were labeled with PKH67 (green). They were tracked under a fluorescence microscope every 30 min for a period of 5 days while we selected the images at 12^th, 24^th, 48^th, and 60^th h in a, or each day for display in b. White circles highlight NK92 cells that infiltrated to tumor region. c, Flow cytometry gating strategy of SPON2⁺ NK92 cells in outer and inner circles of 3D bio-printed models. This gating strategy was used to identify SPON2⁺ NK92 cells that infiltrated to the inner circle or stayed at the outer circle of 3D-printed HCC models. d, Flow cytometry gating strategy to compare the expressions of IFN-γ and Perforin in SPON2⁺ NK92 cells and SPON2⁻ NK92 cells. This gating strategy was used to evaluate IFN-γ and Perforin expressions in SPON2⁺ and SPON2⁻ NK92 cells after 3 h of PMA and ionomycin stimulation. Numbers in the drawn gates indicate cell percentages. e, Proportions of SPON2⁺ NK92 to all the NK92 cells in outer and inner circles of 3D bio-printed models. Left: exemplar gating strategies (squares) for SPON2⁺ NK92 cell selection. Right: we compared proportion values between outer (n = 6 independent experiments) and inner circles (n = 6 independent experiments) in six bio-printed models (P = 0.0017). Paired two-tailed Student’s t-test. f, We measured IFN-γ and Perforin expressions in SPON2⁻ and SPON2⁺ NK92 cells after 3 h of PMA and ionomycin stimulation. The proportions of IFN-γ⁺ NK92 cells (P = 0.018) and Perforin⁺ NK92 cells (P = 0.0002) were compared between SPON2⁻ NK92 (n = 4 cells) and SPON2⁺ NK92 (n = 4 cells) cells. Paired two-tailed Student’s t-test. g, Flow cytometry verification of SPON2 expression in three cell lines. SPON2 expression in SPON2-overexpression NK92 (NK92^OE-SPON2) cells and SPON2-knockdown NK92 (NK92^KD-SPON2) cells relative to empty vector NK92 (NK92^EV) cells. NK92^KD-SPON2 cells were obtained via lentiviral shRNA transfection which achieved a 99% reduction in SPON2 expression. Numbers in the drawn gates indicate cell percentages. h, Representative images depicting the migration of NK92^EV, NK92^OE-SPON2, and NK92^KD-SPON2 toward the tumor region. We documented videos of NK92^EV (left), NK92^OE-SPON2 (middle), and NK92^KD-SPON2 (right; NK92 cells were colored in red) migration towards HepG2 cells (green) during a 12-h period. At the 12^th h, images were captured by fluorescence microscopy (see Supplementary Videos 2–4). In these migration assays comparing NK92^OE-SPON2 and NK92^KD-SPON2 cells to NK92^EV cells, NK92^OE-SPON2 cells migrated to HepG2-cell-rich area within 12 h, while NK92^EV and NK92^KD-SPON2 cells did not. i, IFN-γ and Perforin expressions in NK92^EV cells and NK92^OE-SPON2 cells. The left two subfigures show the distributions of cells with different fluorescence intensities: IFN-γ (left) and Perforin (right) measurement. We use the maximal intensity value of ISO (vertical black dashed lines) as the threshold to call IFN-γ + (or Perforin⁺) and IFN-γ⁻ (or Perforin⁻). We quantified the proportions of IFN-γ⁺ (or Perforin⁺) NK92 cells in NK92^EV and NK92^OE-SPON2 cells. In the right two subfigures, we compared these proportion values between NK92^EV cells (n = 5) and NK92^OE-SPON2 cells (n = 5) in three flow cytometry experiments (P = 8.0 × 10⁻⁴, P = 0.12), using paired two-tailed Student’s t-tests. Statistical significance: *P < 0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001, and ns—not significant.

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