Figure 4.

Single injection of super-charged NK-cells with/without feeding with AJ2 inhibited tumor growth due to differentiation of tumors in hu-BLT mice. Implantation of tumor cells in the pancreas and tail vein injection of super-charged NK cells and feeding with AJ2 (5 × 10⁹) were carried out as depicted in the figure (A), and disease progression was monitored. Mice were sacrificed, and pancreatic tumors were harvested and weighed (n = 4/each experimental condition) (B). Implantation of tumor cells in the pancreas and tail vein injection of super-charged NK cells were carried out as depicted in Figure 4A, and at the time of sacrifice mice were bled and the levels of IFN-γ in the serum were determined using multiplex array (n = 3) (C). Procedures were carried out as depicted in Figure 4A, Figure S3A and Figure S4C before pancreatic tumors were harvested and weighed (n = 3) (D). Upon sacrifice, pancreatic tumors were harvested and single cell suspensions were prepared as described in Materials and Methods. The same numbers of pancreatic tumor cells from each mouse were cultured, and the pictures of cultured tumors were taken on day 7. One of the four representative experiments is shown in the figure (E). Procedures were carried out as described in Figure 4A using injections of allogeneic or autologous super-charged NK cells. Pancreatic tumors were resected and single-cell suspensions were prepared and tumor growth were assessed (n = 9 to 12/each experimental condition) using identical numbers of tumors cultured from each mouse tumor (F). Hu-BLT mice were implanted with MP2 tumors and injected with NK cells or implanted with NK-differentiated tumors as described in Figure 4A, and Supplementary Figures S3A and S4C. At the end of the experiment pancreatic tumors were harvested and tumor growth was assessed on days 7, 11 and 14, and on day 7 attached tumors from each well were counted and equal numbers of tumors from each group were re-cultured and tumor growth in each well was determined every 3 days (n = 12/each experimental condition, one representative experiment is shown in the figure) (G). Hu-BLT mice were implanted with tumors and injected with super-charged NK cells, as described in Figure 4A. Tumors were resected, and single cell cultures were prepared and cultured for 7 days, after which percentages of human CD45, CD94, CD56, NKG2D, and DNAM within the tumors were determined after staining with antibodies, followed by flow cytometric analysis (H,I, Supplementary Figure S3C,D). Procedures were carried out as described in Figure 4A and Supplementary Figure S3A. Pancreata were removed, and single cell cultures were prepared and equal numbers of pancreatic cells were cultured until day 7 or 11 and the levels of IFN-γ (J) and IL-6 (K) were determined in culture supernatants (n = 4/each experimental condition). Hu-BLT mice were implanted with MP2 tumors and injected with NK cells and fed with AJ2 as described in Figure 4A. Upon sacrifice, tumors were resected, and single cell cultures were prepared, and equal numbers of tumors were cultured on day 7 or 11 and the levels of IFN-γ were determined in culture supernatants (n = 2/each experimental condition) (L). Procedures were carried out as described in Figure 4A. Tumors were resected, and single cell cultures were prepared and surface expressions of MHC-class I, B7H1 and CD54 were determined on tumors after culture (n = 4/each experimental condition) (M). NK cells (1 × 10⁶ cells/mL) from healthy individuals were treated with IL-2 (1000 U/mL) for 18–24 h before they were added to ⁵¹Cr labeled tumors obtained from mice implanted with different tumors and/or injected with NK cells as described in Figure 4A at various effector to target ratios. NK cell mediated cytotoxicity was determined using 4 h ⁵¹Cr release assay, and the lytic units 30/10⁶ cells were determined using inverse number of NK cells required to lyse 30% of the tumor-cells × 100 (n = 3/each experimental condition) (N,O). The following symbols represent the levels of statistical significance within each analysis, *** (p-value < 0.001), ** (p-value 0.001–0.01), * (p-value 0.01–0.05)