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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: Clin Cancer Res. 2021 Oct 1;27(19):5376–5388. doi: 10.1158/1078-0432.CCR-21-1621

Figure 4. M2 overcomes downregulation of CD38 in MM cells triggered by IL6 and BMSC supernatant, and further enhances Dara-induced lysis of drug-resistant patient MM cells.

Figure 4.

A IMiDs-resistant H929(R), ANBL6, and its paired bortezomib-resistant ANBL6-BR cells were incubated with M2 for 1d. IL6 (2 ng/ml) was also added in ANBL6 and ANBL6-BR cultures. Cell lysates were made for immunoblotting to determine levels of CD38 and indicated proteins, with GAPDH as a loading control. B BMSCs from patients were cultured for 3d in the same culture medium as MM cells. Culture supernatants were then harvested, passed through 0.2 μm filters, mixed with 50 % of control culture medium, and used as BMSC culture supernatant (BMSC-sup). H929(R) cells were pretreated with ctrl or M2 (0.1, 0.5 µg/ml) for 1d in the absence or presence of IL6 (2 ng/ml) or BMSC-sup for 2d. C H929(R) target cells, pretreated with M2 or ctrl for 1d, were co-cultured with NK cells (n = 3 normal donors) in Dara-induced ADCC assay in the absence or presence of BMSC-sup from cultures of 2 patient samples. Using quantitative FC analysis, the % H929(R) cell lysis was determined. D Dara-induced lysis of M2- vs ctrl-treated RPMI8226 cells was assayed in the presence of BMSCs (n = 3). Patient PBMCs (n = 3) were used as effector cells. E Using FC analysis, CD38 surface expression was analyzed on CD138+Aqua-/Annexin V- viable patient cells treated with 1 µg/ml M2 vs ctrl for 2d (RRMM, n = 24). Two representative samples are shown on the right. F BM mononuclear cells (BMMCs) from a RRMM patient were treated with M2 vs ctrl (1 µg/ml) for 1d, followed by co-culture with PBMC from the same patient in the presence of Dara for 2d. Using quantitative FC analysis, the % lysis of CD138+ cells was determined. BMMCs from Dara-resistant patients (n = 3) were treated with M2 followed by assessment of CD38 surface expression on viable CD138+ cells (G); samples after M2 (1 µg/ml) treatment for 1d were used to determine % Dara-induced autologous patient MM lysis in the presence of paired PBMCs (H). Three independent experiments (C-D) were done using NK cells from 3 individuals in triplicates at each condition. All results are shown as means ± SDs (error bars). *P < .05, **P < .01, ***P < .001, ****P < .0001