Boosting Natural Killer Cell-Mediated Targeting of Sarcoma Through DNAM-1 and NKG2D

Abstract

Sarcomas are malignancies of mesenchymal origin that occur in bone and soft tissues. Many are chemo- and radiotherapy resistant, thus conventional treatments fail to increase overall survival. Natural Killer (NK) cells exert anti-tumor activity upon detection of a complex array of tumor ligands, but this has not been thoroughly explored in the context of sarcoma immunotherapy. In this study, we investigated the NK cell receptor/ligand immune profile of primary human sarcoma explants. Analysis of tumors from 32 sarcoma patients identified the proliferative marker PCNA and DNAM-1 ligands CD112 and/or CD155 as commonly expressed antigens that could be efficiently targeted by genetically modified (GM) NK cells. Despite the strong expression of CD112 and CD155 on sarcoma cells, characterization of freshly dissociated sarcomas revealed a general decrease in tumor-infiltrating NK cells compared to the periphery, suggesting a defect in the endogenous NK cell response. We also applied a functional screening approach to identify relevant NK cell receptor/ligand interactions that induce efficient anti-tumor responses using a panel NK-92 cell lines GM to over-express 12 different activating receptors. Using GM NK-92 cells against primary sarcoma explants (n = 12) revealed that DNAM-1 over-expression on NK-92 cells led to efficient degranulation against all tested explants (n = 12). Additionally, NKG2D over-expression showed enhanced responses against 10 out of 12 explants. These results show that DNAM-1⁺ or NKG2D⁺ GM NK-92 cells may be an efficient approach in targeting sarcomas. The degranulation capacity of GM NK-92 cell lines was also tested against various established tumor cell lines, including neuroblastoma, Schwannoma, melanoma, myeloma, leukemia, prostate, pancreatic, colon, and lung cancer. Enhanced degranulation of DNAM-1⁺ or NKG2D⁺ GM NK-92 cells was observed against the majority of tumor cell lines tested. In conclusion, DNAM-1 or NKG2D over-expression elicited a dynamic increase in NK cell degranulation against all sarcoma explants and cancer cell lines tested, including those that failed to induce a notable response in WT NK-92 cells. These results support the broad therapeutic potential of DNAM-1⁺ or NKG2D⁺ GM NK-92 cells and GM human NK cells for the treatment of sarcomas and other malignancies.

Keywords: DNAM-1 (CD226); NK-92 cell line; NKG2D (Natural killer group 2 member D); cancer immunology; cancer immunotherapy; natural killer (NK) cell; sarcoma.

Figure 1

Characterization of peripheral and tumor-infiltrating T and NK cell populations from freshly isolated sarcoma patient material. (A) Representative gating strategy and (B) percentages of leukocytes: CD45⁺, T_{helper/regulatory} cells: CD3⁺CD4⁺, T_cytotoxic cells: CD3⁺CD8⁺ and NK cells: CD3⁻CD56⁺ among fresh PBMC and TIL of sarcoma patients (n = 14). The Live/Dead exclusion gate also includes markers CD14-V500 and CD19-V500 as a dump channel (C) Percentages of the Q1: CD16⁺KIR⁻, Q2: CD16⁺KIR⁺, Q3: CD16⁻KIR⁺, and Q4: CD16⁻KIR⁻ of NK cells from PBMC and TIL of sarcoma patients from (C) (n = 14). (D) Relative Median Fluorescence Intensity of DNAM-1 and NKG2D expression on NK cells in PBMC and TIL of sarcoma patients (n = 14). Statistical analysis was performed by Wilcoxon matched pairs signed rank non-parametric t-test and the Grubb's outlier test. In (B,C) the outlier is marked with a red square and the p-value is based on statistical analysis upon exclusion of the outlier.

Figure 2

Lymphocyte ligand expression profile of fresh and in vitro propagated sarcoma explants. (A) Representative gating strategy for flow cytometry-based analysis of lymphocyte ligand surface expression on in vitro propagated sarcoma explants. In the case of fresh sarcoma explants, CD45-BV510 is included in the same channel as Live/Dead Aqua as an exclusion marker. (B,C) Fold MFI of lymphocyte ligands on fresh sarcoma explants. Normalization: (sample MFI – unstained MFI)/unstained MFI (n = 13). (D,E) Fold MFI of lymphocyte ligands on in vitro propagated sarcoma explants. Normalization: (sample MFI – unstained MFI)/unstained MFI (n = 32).

Figure 3

GM NK-92 cell-based screening platform tested against 12 different primary sarcoma explants. WT or GM NK-92 cells were co-cultured with target cells at 1:1 (E:T) ratio for 4 h. %CD56⁺CD107a⁺ NK-92 cells were analyzed by flow cytometry. PMA and ionomycin (PMA/IO) were used as positive stimulators of degranulation and K562 as the validated target of NK-92 cells. (A) Generation of GM NK-92 cells and gating strategy for all degranulation flow cytometry analysis. (B) Dot plots and heatmap showing normalized %CD56⁺CD107a⁺ WT or GM NK-92 cells against each sarcoma explant and sarcoma cell lines Saos-2 and U-2 OS (PMA/IO responses for each GM NK-92 cell line was set as 100% for the normalization of the data; results from one representative experiment, plotted as means of technical replicates).

Figure 4

Enhanced degranulation of NKG2D⁺ and DNAM-1⁺ GM NK-92 cells against selected sarcoma explants and Saos-2 and U-2 OS cell lines is receptor mediated. CD155, DNAM-1, or NKG2D blocking was done 15 min prior to degranulation set up. The assay was set as previously described, at E:T ratio 1:1. (A) Anti-CD155 on tumors and anti-DNAM-1 blocking of WT or DNAM-1⁺ GM NK-92 cells against primary sarcoma explants and (B) anti-DNAM-1 blocking of WT or DNAM-1⁺ GM NK-92 cells against Saos-2 and U-2 OS cell lines. (C) Anti-NKG2D blocking of NKG2D⁺ GM NK-92 cells against primary sarcoma explants and (D) anti-NKG2D blocking of WT or NKG2D⁺ GM NK-92 cells against Saos-2 and U-2 OS cell lines (results from two independent experiments, means plotted with error bars indicating SD). (E) Comparison of degranulation by DNAM-1 or NKG2D GM NK-92 cells with NK-92 cells co-expressing both receptors against Saos-2 and U-2 OS cells (^**** indicates p < 0.0001 with 2-way ANOVA analysis).

Figure 5

Evaluation of NK cell cytotoxicity. (A) Representative results from cytotoxicity assays on Saos-2 (left panel) and U-2 OS (middle panel) cell lines using the xCelligence RTCA platform. Calculation of cytotoxic activity after 4 h of co-culture from two independent experiments (right panel) shows enhanced cytotoxic activity by GM NK cells. (B) Live tumor cells were loaded with Calcein-AM dye and co-cultured with effector cells at a 10:1 E/T ratio and incubated for 4 h. Live tumor cells were identified as Hoescht positive nuclei bounded by a Calcein Red positive cytoplasmic border. Apoptotic bodies were size excluded from the analysis. Tumor cell viability was assessed by determining the average fluorescent intensity (AFI) of individual tumor cells within each well. Percent viability was calculated by comparing the AFI of each condition to non-treated controls. Results are reported as the mean viability of two independent experiments, with three technical replicates per experiment and four fields of view per well. Results were analyzed using a two-way ANOVA with Tukey's post-hoc analysis in GraphPad Prism. DNAM-1⁺ NK-92 cells were found to significantly reduce the viability of primary sarcoma patient tumor cells, p < 0.0001 (HTT12, HTT26); p = 0.0018 (HTT25).

Figure 6

TCGA analysis of 259 Sarcoma patients reveals high expression of DNAM-1 and NKG2D ligands are associated with poor survival. (A) Kaplan-Meier survival estimation analysis results from 259 TCGA Sarcoma TPM values based on expression of DNAM-1 ligands (Nectin 2 or CD112, PVR or CD155) and NKG2D ligands (MICA, MICB, ULPB1, ULPB2, ULBP3, RAET1E, or ULBP4 and RAET1G or ULBP5). Data from 20th to 80th percentile was compared. (B) Log2 TPM value of ligands of NKG2D and DNAM-1 from TCGA Sarcoma expression data.

Figure 7

Cancer cell lines expressing CD112 and/or CD155 are targeted by DNAM-1⁺ NK-92 cells. (A) Degranulation response of all GM NK-92 cells and (B) of WT or DNAM-1⁺ NK-92 cells in 4 h at 1:1 (E:T) with indicated cell lines were normalized according to the PMA/ionomycin response of effectors in each run [A: a single representative, B: Means of two independent experiments, run in duplicates are depicted with error bars indicating SEM. All except for RPMI 8226 response give significance (p < 0.0001 with 2-way ANOVA analysis.)] (C) Cancer cell lines were stained for surface expression of CD112 and CD155 and analyzed by flow cytometry (dashed line: unstained, filled histogram: stained). ns, not significant.