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B.A. Williams et al.
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Patient sample ID
Effector:Target Ratio
Figure 1. Chromium release assay of NK- 92 against primary acute myeloid leukemia (AML) samples. (A) Four freshly thawed primary AML blast samples were labeled with 100 mCi of Na251CrO4 prior to treatment with NK-92 at four E:T ratios. (B) AML blast sample 080078 was tested in a separate experiment at four E:T ratios with and without calcium chelator EGTA 4 (mM) and MgCl2 (3 mM). Data are presented as the mean percent lysis of triplicate samples (+/-Standard Deviation) from a representa- tive experiment carried out three times.
killed in a dose-dependent manner and killing was abro- gated in the presence of the calcium chelator EGTA that blocks granule exocytosis (Figure 1B).
To determine the effect of NK-92 on LSCs, we sorted a primary AML sample into CD34+CD38– and CD34+CD38+ fractions for further testing using the CRA. Primary AML-derived CD34+CD38– cells were more sen- sitive to killing than CD34+CD38+ blasts by NK-92 in a 4- hour CRA at E:T ratios of 1:1 (58.9±11.5%, 20.3±1.7%), 5:1 (78.3±9.7%, 43.5+11.1%) and 10:1 (72.9±5.6%, 38.5±2.4%); this difference was not significant at a 25:1 E:T ratio (Figure 2A).
To test the effect of NK-92 against LSCs relative to bulk tumor, we compared the CRA with a methylcellulose cytotoxicity assay (MCA) designed to measure the killing of clonogenic primary AML cells during the 4-hour co- incubation. We also employed a control to correct for the effect of NK-92 against leukemia targets during the 4- week exposure in methylcellulose by enumerating colonies that arose from infusing NK-92 and targets in
methylcellulose without prior incubation. The MCA showed that NK-92 at a 25:1 E:T eliminated clonogenic growth of most primary AML blast samples yielding % colony inhibition values of: 86.3±2.3%, 98.4±2.8%, 100±0% and 100±0%, demonstrating much higher cyto- toxicity than obtained with the CRA, which was per- formed on the same day (Figure 2B and C).
To determine if the difference in cytotoxicity measured by the CRA and MCA was not simply due to method- ological reasons, we screened additional cell line targets using this methodology. While OCI/AML3 clonogenic cells were more sensitive to NK-92 than bulk tumor, OCI/AML2 bulk and clonogenic cells were equivalently sensitive to NK-92 (Online Supplementary Figure S3). OCI/AML2 was the only target tested which did not have a differential cytotoxicity measured by the CRA and MCA. This demonstrates that the enhanced cytotoxicity measured in the MCA compared with the CRA is, for most targets, not intrinsically related to the method of data comparison.
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haematologica | 2018; 103(10)