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. Author manuscript; available in PMC: 2025 Jan 14.
Published in final edited form as: Cancer Immunol Res. 2025 Mar 4;13(3):430–446. doi: 10.1158/2326-6066.CIR-24-0189

Figure 2. Quantification of NK cell-mediated cancer immunoediting in vitro.

Figure 2

(A) Schematic representation of the proposed model. Scheme was created in BioRender. Kovar, H. (2023) BioRender.com/j11m245. (B) Upon long-term co-culture of B-ALL and NK cells, we hypothesised that each tumour cell clone would fall into one of the following categories: The abundance of a clone can be significantly higher (primary resistant) or lower (eliminated), unchanged (static) or show a high variability (secondary resistant) upon NK cell co-culture. Variability is defined as v = log2(max(x + 1)/min(x + 1)), where x is a vector of normalised counts across samples. A rule-based decision tree was designed to discriminate the four categories depicted in (A). (C) The percentage of the cell clones in each group on day 14 is depicted for cell lines A, B, C and D. Shown are the mean values of n=2-3 independent experiments, where each individual data point represents the mean value (n=3 wells, sequenced in duplicates) of one experiment. (D) The heatmap shows the normalised abundance of barcodes (norm. BC abundance) of cell line A from one representative experiment (n=2-4 cell lines, 4 independent experiments). Each subcolumn (n=3) in the figure represents a sample (well), and each row represents a barcode. The barcodes were divided into the four pre-defined groups according to the criteria defined in (B). The Shannon diversity index (shown above the heatmap) serves as measure of barcode diversity and drops significantly after 14 days of NK cell co-culture. The histogram on the left shows the average abundance of the barcoded cell clone in the B-ALL only samples on day 4. (E) The bubble plot depicts the normalised abundance of secondary resistant clones in B-ALL samples (day 4) compared to B-ALL + NK samples (day 14). The x-axis shows the 3 individual wells of each condition, while each row on the y-axis shows an individual tumour cell clone. The size and colour of the bubbles indicate the normalised barcode abundance. Shown is the same cell line and experiment as in (D). (F) Comparing 2 independent experiments in cell line A as shown in (D&E), the Euler diagrams highlight a high overlap of eliminated, primary resistant, and static cell clones. In contrast, only 2 secondary resistant clones were shared between both independent experiments.