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. 2021 Oct 29;13(21):5446. doi: 10.3390/cancers13215446

Figure 6.

Figure 6

Activity of the bispecific C-7 VHH using metastatic CRC patient-derived PBMC and dissociated tumor samples. (A) Degranulation of NK cells (defined as CD45+CD56+CD3− cells) and (B) cytotoxicity of A431 tumor cells after a 4-h (degranulation) and 24-h (cytotoxicity) co-culture of monocyte-depleted PBMC derived from patients with stage II/III CRC pre-activated overnight with IL-2 and IL-15 ± A431 (EGFR++) ±100 nM C-7. E:T ratio 4:1. Degranulation was determined by assessing the percentage of NK cells expressing CD107a+. Cytotoxicity was determined by assessing the relative percentage of living tumor cells compared to the tumor-alone control. n = 5 (A), n = 4 (B). (C) EGFR MFI of Epcam+CD45− tumor cells in dissociated CRC peritoneal metastatic lesions (n = 15) and EpcamdimCD45− epithelial cells, in (non-malignant) peritoneal tissue (n = 3). CD56+CD3− (D) and CD16+CD56+CD3− (E) percentage of CD45+ cells in dissociated CRC peritoneal metastatic lesions, n = 6. (F) Absolute number of Epcam+CD45− tumor cells from dissociated CRC peritoneal metastatic lesions cultured ± autologous PBMC (E:T ratio 5:1) ±100 nM C-7 for 1, 3, and 7 days. n = 8. The significance level refers to the conditions: “T+PBMC” versus “T+PBMC+C-7”. (G) Absolute number of EpcamdimCD45− cells from dissociated normal (non-malignant) peritoneal tissue of patients with metastatic CRC ± autologous PBMC (E:T ratio 5:1) ±100 nM C-7, incubated for 1 and 3 days. n = 3. (H) Cytometry Bead Array performed on supernatant of dissociated CRC peritoneal metastases cultured for 24 h ± autologous PBMC (E:T ratio 5:1) ± 100 nM C-7. n = 9. The data are presented as mean ± SEM. Significance is presented as p < 0.05 *, <0.01 **, <0.001 ***. No p-values are mentioned in case p > 0.05. p-values are determined by two-tailed paired t-test (A,B); Mann–Whitney test (C); two-way ANOVA with Dunnett’s multiple comparison test (F,G); one-way ANOVA with Dunnett’s multiple comparison test (H: IL-6); Friedman test with Dunn’s multiple comparison test (H: IFNγ, TNF, CXCL10, IL-10). Abbreviations: T = tumor, C-7 = C21-7D12 bispecific VHH, PBMC = peripheral blood mononuclear cells.