Chemotherapy-resistant osteosarcoma is highly susceptible to IL-15-activated allogeneic and autologous NK cells

Abstract

High-grade osteosarcoma occurs predominantly in adolescents and young adults and has an overall survival rate of about 60%, despite chemotherapy and surgery. Therefore, novel treatment modalities are needed to prevent or treat recurrent disease. Natural killer (NK) cells are lymphocytes with cytotoxic activity toward virus-infected or malignant cells. We explored the feasibility of autologous and allogeneic NK cell-mediated therapies for chemotherapy-resistant and chemotherapy-sensitive high-grade osteosarcoma. The expression by osteosarcoma cells of ligands for activating NK cell receptors was studied in vitro and in vivo, and their contribution to NK cell-mediated cytolysis was studied by specific antibody blockade. Chromium release cytotoxicity assays revealed chemotherapy-sensitive and chemotherapy-resistant osteosarcoma cell lines and osteosarcoma primary cultures to be sensitive to NK cell-mediated cytolysis. Cytolytic activity was strongly enhanced by IL-15 activation and was dependent on DNAM-1 and NKG2D pathways. Autologous and allogeneic activated NK cells lysed osteosarcoma primary cultures equally well. Osteosarcoma patient-derived NK cells were functionally and phenotypically unimpaired. In conclusion, osteosarcoma cells, including chemoresistant variants, are highly susceptible to lysis by IL-15-induced NK cells from both allogeneic and autologous origin. Our data support the exploitation of NK cells or NK cell-activating agents in patients with high-grade osteosarcoma.

Fig. 1

Osteosarcoma cells were sensitive to lysis by freshly isolated NK cells (solid lines) and NK cells cultured in IL-15 for 2 weeks (dashed lines). a examples of percentage of specific lysis are shown for the osteosarcoma cell lines SAOS-2 (filled circle), 143B/HOS (open circle), IOR/OS-14 (filled square), and ZK-58 (open square). Cell lines were incubated with increasing numbers of NK cells (E:T; effector to target ratio). Error bars represent standard error of the mean lysis of a representative experiment performed in triplicate. b mean percentage of specific lysis by unstimulated (white bars) and IL-15-activated (black bars) NK cells of the osteosarcoma cell lines 143B/HOS, SJSA-1, OHS, U2-OS, SAOS-2, IOR/OS-14, HOS, and ZK-58 at an effector to target ratio of 10:1. Error bars represent standard error of the mean of independent experiments using different healthy donor NK cells. Numbers in the bars represent number of experiments. K562 and an EBV-transformed B-LCL (“EBV”) were used as positive and negative controls, respectively. Mann–Whitney U test was done comparing IL-15-activated NK cells with unstimulated NK cells for each cell line; P-value <0.05 noted as *; <0.01 as **; <0.001 as ***

Fig. 2

a Examples of immunohistochemical staining of ligands for the activating receptors NKG2D (MICA) and DNAM-1 (CD112 and CD155) and of β2-microglobulin, HLA-A (HCA2), and HLA-B/C (HC10) on osteosarcoma samples. b Overview of the results of immunohistochemical stainings on pre-chemotherapy and post-chemotherapy samples of the primary tumor as well as metastatic osteosarcoma tissue. Expression levels of CD112 and CD155 but not the other ligands decreased significantly upon chemotherapy treatment (P-value Kruskal–Wallis test <0.001 as ***). c Example of flow cytometry plots for MICA, CD112, CD155, and HLA class I for the osteosarcoma cell line IOR/OS-14; isotype-matched control staining is shown in gray

Fig. 3

a cytolysis of U2-OS by unstimulated (solid lines) and IL-15-activated (dashed lines) NK cells was almost completely abrogated when the NK cells were pre-incubated with both anti (α)-DNAM-1- and α-NKG2D-blocking antibodies (filled square vs. open square). Unstimulated NK cells were most dependent on DNAM-1 (open circle) signaling, whereas activated NK cells were most dependent on NKG2D (filled circle). Error bars represent standard error of the mean lysis of experiment performed in triplicate. Similar results were obtained for SAOS-2, HOS, and ZK-58 using unstimulated (b) and IL-15-activated NK cells (c). Bars represent mean lysis in at least three independent experiments using healthy donor NK cells; error bars represent standard error of the mean. Friedman test, Dunn’s post-test compared to non-blocked; P-value <0.05 noted as *; <0.01 as **; <0.001 as ***

Fig. 4

a Lysis of U2-OS variants resistant to doxorubicin (DX), methotrexate (MTX), or cisplatinum (CDDP) was comparable to lysis of the parental control. The SAOS-2 CDDP-resistant line was less sensitive to lysis by unstimulated NK cells than its parental control (Friedman test P-value = 0.001 and Dunn’s post-test compared to parental cell line P-value <0.001). NK cell activation (“IL-15+”) increased lysis in all cases. Error bars represent standard error of the mean lysis in at least six independent experiments. b SAOS-2 expressed the death receptor CD95 (Fas; black line), whereas expression was lost in the cisplatinum-resistant variant of SAOS-2 (dashed line). Isotype-matched control staining is shown in gray. c the granzyme/perforin pathway was the main cytotoxic pathway by which NK cells lysed SAOS-2 and its chemotherapy-resistant variants, as shown by inhibition of granule exocytosis by concanamycin A. Blocking Fas resulted in a further decrease in the lysis of SAOS-2, but not of the chemotherapy-resistant variants. Bars represent mean lysis of experiment performed in triplicate; error bars represent standard error of the mean. Similar results were obtained using NK cells from another donor

Fig. 5

a NKG2D level was similar in unstimulated NK cells of newly diagnosed osteosarcoma patients and healthy controls. Following culture for 3 days in IL15, there was a larger increase in the expression level of NKG2D in NK cells of patients. b Unstimulated PBMCs of 12 of 22 patients and 16 of 23 healthy controls and IL-15-activated PBMCs of 19 of 22 patients and 17 of 23 controls were available for functional testing. NK cells of newly diagnosed osteosarcoma patients and of healthy controls lysed HOS at similar levels. Following IL-15 activation, NK cells of osteosarcoma patients showed a larger increase in cytotoxic activity than NK cells of healthy donors. c cytotoxicity of IL-15-activated NK cells correlated with level of NKG2D expression on the CD56dim subset (Pearson correlation coefficient). d primary osteosarcoma cell cultures were tested for sensitivity to lysis by autologous (open circle) and allogeneic (closed circle) NK cells. NK cells were unstimulated (solid lines) or 3 days IL-15 activated (dashed lines). Autologous IL-15-activated NK cells were available for all patients except L2792. One-way analysis of variance (ANOVA) P-value <0.0001 for a, b, and c. Bonferroni’s multiple comparison post-test; P-value <0.05 noted as *; <0.01 as **; <0.001 as ***