Carfilzomib modulates tumor microenvironment to potentiate immune checkpoint therapy for cancer (2021)
Figure 2

Figure 2. Carfilzomib reprogrammed M2 macrophages into M1-like population

A. The most effective one among the candidate compounds, Carfilzomib, significantly promotes the expression of M1 macrophage markers, as well as reduces the expression of M2 macrophage markers. BMDMs were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM). RNA was extracted from cells 6 hours after stimulation and the expression of M1 (Il-6/Inos) or M2 (Cd206/Arg1) macrophage markers were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. ***p < 0.001 (student's t-test).

B. Carfilzomib significantly promotes the secretion of IL-6 and TNFα in M2 macrophages. BMDMs were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (500 nM). The secretion of IL-6 and TNFα were detected through ELISA 24 hours after stimulation. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. **p < 0.01, ***p < 0.001 (student's t-test).

C. Carfilzomib significantly promotes the expression of membrane protein CD86, CD80, MHC-II and reduces the expression of CD206. BMDMs were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (500 nM). The representative histogram of CD86, CD80, MHC-II and CD206 expression was analyzed through flow cytometry 12 hours after stimulation. The gating of CD86+, CD80+, CD206+ and MHC-II+ population was determined against those of isotype-matched staining control.

D. Statistics for proportion of CD86, CD80, MHC-II or CD206 positive cells in BMDMs. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test).

E. Carfilzomib promotes the phagocytosis of macrophages. BMDMs were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (500 nM) for 12 hours. After starving for 2 hours and stained with Red membrane dye, BMDMs (red) were incubated with L1210-GFP cells (green) in serum-free medium for another 2 hours. Phagocytosis effect was observed and photographed under fluorescence microscope. Scale bars, 50 μm (up), 20 μm (down). The yellow arrows indicated L1210 that is phagocytosed by macrophages.

F. Statistics represent the number of phagocytosis L1210 in 100 macrophages. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).

G, H. Phagocytosis is measured by flow cytometry. After co-incubation, BMDMs were stained with fluorochrome-conjugated anti-CD11B antibody and analyzed through Flow cytometry. Phagocytosing efficiency was determined as the percentage of CD11B and GFP double positive population. Statistics represent the percentage of phagocytosing against total macrophage population. Statistics represent the percentage of phagocytosing against total macrophage population and data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).

I-L. Ci-M1 drives proliferation of CD8+ or CD4+ T cells. BMDMs treated as indicated were transfected with 10 μg/mL of OVA257-264 peptide or OVA323-339 peptide for 1 hours. BMDMs were then washed and co-incubated with CFSE-labeled OT-I or OT-II cells respectively. After 72 hours, OT-I (I) or OT-II (K) cells were stained with anti-CD8a or anti-CD4 fluorochrome-conjugated antibodies and CFSE was detected through flow cytometry to evaluate the dilution of CFSE in T cells. Statistics represent the percentage of divided T cells. (J, L) are statistics analysis of Figure 2I and 2K. The data are means ± SD of three independent experiments. *p < 0.05 (student's t-test).

M. Carfilzomib promotes the M1-polarization of Tumor associated macrophages in vitro. BMDMs were cultured with tumor culture supernatant (TSN) produced by L1210 cells, followed by activation with DMSO or Carfilzomib (1 μM). RNA was extracted from cells and the expression of Il-1β, Il-6, Inos were quantified through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. **p < 0.01, ***p < 0.001 (student's t-test).