Journal: Immunotherapy Advances

Article Title: SMAC mimetics induce human macrophages to phagocytose live cancer cells

doi: 10.1093/immadv/ltaf026

Figure Lengend Snippet: SMAC-mimetics induce phagocytosis in human macrophages. (A) Representative flow cytometry plots from a single donor showing the CTV⁺CD45⁺ phagocytosis population within the total CD45⁺ macrophage population. (B) Macrophages from n =8 healthy donors were treated with the 500 nM of the indicated SMAC mimetics for 24 hours prior to co-culture with CellTrace Violet labeled MDA-MB-231 breast cancer cells. Phagocytosis was assessed by flow cytometry after 18 hours of co-culture. Results from each donor were normalized to the vehicle control from that donor. Error bars are SD. Statistical significance was determined using a pairwise one-way ANOVA with Sidak’s multiple comparison. (C) Immunoblot analysis of macrophage lysates treated with SMAC-mimetics (500 nM) for 24 hours. Blots were probed for cIAP1, XIAP, and GAPDH. (D) The frequency of macrophages recovered after 18 hours co-culture with tumor cells under each of the indicated treatment conditions. (E) Macrophages from n = 5 healthy donors were treated with the indicated concentrations of LCL161 for 24 hours prior to co-culture with CellTrace Violet labeled MDA-MB-231 breast cancer cells. Phagocytosis was assessed by flow cytometry after 18 hours of co-culture. Results from each donor were normalized to the vehicle control from that donor. Error bars are SD. Statistical significance was determined using an ordinary one-way ANOVA. (F) Immunoblot of protein lysates from macrophages treated for 24 hours with the indicated concentrations of LCL161.

Article Snippet: The next day, tumor cells were collected and labelled with CellTrace Violet (CTV) (Fisher Scientific, Cat# C34557 ) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Co-Culture Assay, Labeling, Control, Comparison, Western Blot

Journal: Immunotherapy Advances

Article Title: SMAC mimetics induce human macrophages to phagocytose live cancer cells

doi: 10.1093/immadv/ltaf026

Figure Lengend Snippet: Autocrine TNFa sustains the phagocytosis phenotype. (A) Healthy donor macrophages were treated with 500 nM LCL161 and 50 ng/mL IFNg or vehicle control. TNFa was measured by ELISA of 24 hour culture supernatants and reported as absorbance values. Error bars are SD. (B) Healthy donor macrophages from n = 3 independent donors were treated with DMSO, LCL161, or LCL161+IFNg for 18 hours of co-culture with CellTrace Violet labeled PANC-1 pancreatic cancer cells with or without TNFa blocking antibodies (1 mg/mL). Phagocytosis was assessed by flow cytometry after 24 hours of co-culture. Error bars are SD. Statistical significance was determined using a pairwise one-way ANOVA with Sidak’s multiple comparisons assuming sphericity. (C) Graphical overview of the follow-up experiment in which the antibody was added both during pre-treatment with DMSO, LCL161, or LCL161+IFNg for 24 hours, and during co-culture with CellTrace Violet labeled PANC-1 cells for 18 hours. Phagocytosis was again assessed by flow cytometry. (D) Phagocytic index of macrophages from n = 6 independent healthy donors. For each donor, data were normalized to vehicle + isotype. The Wilcoxon matched-pairs signed rank test was used to determine statistical significance. (E) Bone marrow-derived macrophages from a WT- and TNFa KO mouse were pre-treated with DMSO, LCL161, or LCL161+IFNg for 24 hours. Macrophages were co-cultured with 6694c2 mouse pancreatic cancer cells for an additional 18 hours. Flow cytometry was used to measure phagocytosis. Error bars are SD. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: The next day, tumor cells were collected and labelled with CellTrace Violet (CTV) (Fisher Scientific, Cat# C34557 ) according to the manufacturer’s instructions.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Labeling, Blocking Assay, Flow Cytometry, Derivative Assay, Cell Culture