Journal: Smart Medicine

Article Title: Magnetic Reprogramming of Macrophages Stimulates Phagocytosis of Breast Cancer Cells via a TRPC1‐STING Inflammatory Axis

doi: 10.1002/smmd.70038

Figure Lengend Snippet: PEMF (3 mT, 10 min) activates the STING pathway in RAW264.7 macrophages. (A) Representative western blots and (B) fold change quantification of phosphorylated (i) STAT1, (ii) NF‐κB (s536), (iii) NF‐κB (s468), (iv) IκB‐α, (v) IKK, (vi) TBK1 and (vii) IRF3 in a time‐course study after PEMF exposure. ( n = 3). (C) Representative western blot run on non‐reducing SDS‐PAGE and (D) fold change quantification of STING dimers normalized to STING monomer. ( n = 3). (E) Representative western blots and (F) fold change quantification of phosphorylated NF‐κB 4 h after PEMF exposure in cells pretreated with inhibitors of NLRP3 (0.5 μM MCC950), STING (2 μM C176), TBK1 (100 μM Amlexanox), and ROS (5 mM N‐acetylcysteine, NAC) for 1 h ( n = 5). (G) Representative western blots and (H) fold change quantification of (i) STING and (ii) phosphorylated NF‐κB 4 h post PEMF exposure. RAW264.7 macrophages were analyzed 24 h post‐transfection with non‐targeting dsiRNA (NC) or dsiRNA against STING ( n = 3). (I) Representative western blots and (J) fold change quantification of phosphorylated NF‐κB 4 h post PEMF exposure in wildtype and cGAS knock‐out RAW264.7 cells ( n = 3). (K) Representative western blots and (L) fold change quantification of phosphorylated (i) STING, (ii) IRF3, (iii) NF‐κB and (iv) TBK1 in a time‐course study after treatment with 20 μg/mL DMXAA and 10‐min PEMF (3 mT). PEMF exposure was administered immediately after DMXAA treatment ( n = 3). All phosphoprotein levels were normalized to total protein, except for phosphorylated STING and phosphorylated NF‐κB, which were normalized to the loading control. Residual antibody binding to the phosphorylated form of NF‐κB hindered the reprobing of total NF‐κB. Phosphorylated NF‐κB was normalized to the loading control, with total NF‐κB assessed on a separate gel. All samples were normalized to their time/drug matched 0 mT control except in (L) where samples were normalized to 0 mT at 0 h. Dots represent individual biological replicates. Data represent mean ± SEM. Statistical significance was determined by Student's unpaired t ‐test (D), one‐way (H (i)) or two‐way ANOVA (B, F, H (ii), J, L) with Šidák's post hoc test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and ****, p ≤ 0.0001).

Article Snippet: AP‐18 (10 μM), RN‐9893 (1 μM), 5,6‐dimethylxanthenone‐4‐acetic acid (DMXAA) (20 μg/mL), C176 (2 μM), and Amlexanox (100 μM) were purchased from MedChemExpress.

Techniques: Western Blot, SDS Page, Transfection, Knock-Out, Control, Binding Assay

Journal: Smart Medicine

Article Title: Magnetic Reprogramming of Macrophages Stimulates Phagocytosis of Breast Cancer Cells via a TRPC1‐STING Inflammatory Axis

doi: 10.1002/smmd.70038

Figure Lengend Snippet: TRPC1 contributes to STING activation (A) Representative western blots and (B) quantification of phosphorylated NF‐κB 4 h after PEMF exposure in RAW264.7 cells pretreated for 1 h with aminoglycoside antibiotics (gentamicin, streptomycin, neomycin, penicillin‐streptomycin) or TRPC inhibitors (10 μM 2‐APB, 50 μM SKF‐96365). All samples were normalized to their drug‐matched 0 mT control ( n = 5). (C) Representative western blots showing phosphorylated proteins following DMXAA treatment (20 μg/mL) in TRPC‐inhibited macrophages after 1 and 2 h post‐treatment. Bar charts in (D) show quantification of phosphorylated STING, IRF3, and TBK1 at 1 h (i–iii), and of STING, STAT1, and NF‐κB at 2 h (iv–vi). All samples were normalized to the vehicle control ( n = 4). (E) Representative western blots of TRPC1‐silenced macrophages after PEMF exposure and (F) quantification of (i) TRPC1 and (ii) phosphorylated NF‐κB ( n = 3). (G) Representative western blots of STIM1‐silenced macrophages in the presence of STING inhibitor C176, with (H) quantification of (i) STIM1 and (ii) phosphorylated NF‐κB. (I) Normalized Calcium Green absorbance comparing real‐time calcium influx in RAW264.7 cells immediately after PEMF exposure, highlighting the inhibitory effects of 15 min pre‐treatment with Streptomycin, SKF‐96365, Pico145, AP‐18 and RN‐9893 ( n = 3). Phosphoprotein levels were normalized to their respective total protein levels, with the exceptions of phosphorylated STING and phosphorylated NF‐κB, which were normalized to GAPDH/β‐actin loading control. Dots represent individual biological replicates. Data represent mean ± SEM. Statistical significance was determined by one‐way (D, F (i), H (i)) or two‐way (B, F (ii), H (ii), I) ANOVA with Šidák's post hoc test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and ****, p ≤ 0.0001).

Article Snippet: AP‐18 (10 μM), RN‐9893 (1 μM), 5,6‐dimethylxanthenone‐4‐acetic acid (DMXAA) (20 μg/mL), C176 (2 μM), and Amlexanox (100 μM) were purchased from MedChemExpress.

Techniques: Activation Assay, Western Blot, Control

Journal: Smart Medicine

Article Title: Magnetic Reprogramming of Macrophages Stimulates Phagocytosis of Breast Cancer Cells via a TRPC1‐STING Inflammatory Axis

doi: 10.1002/smmd.70038

Figure Lengend Snippet: PEMF (3 mT, 10 min) activates STING pathway in 4T1. (A) Representative western blots and (B) fold change quantification of phosphorylated (i) NF‐κB, (ii) IRF3, and (iii) TBK1 in a time‐course study after PEMF exposure ( n = 3). (C) Representative western blots and (D) fold change quantification of phosphorylated NF‐κB 1 h after PEMF‐exposure of 4T1 pre‐treated with STING (2 μM C176) or TRPC1 (streptomycin) inhibitors for 1 h ( n = 3). (E) Representative western blots and (F) fold change quantification of (i) phosphorylated NF‐κB (ii) CD86 and (iii) iNOS in RAW264.7 macrophages after 24 h incubation in PEMF‐conditioned media from 4T1 ( n = 4/3). Phosphoprotein levels were normalized to their respective total protein abundances, except for phosphorylated NF‐κB, which was normalized to the loading control. Residual antibody binding to the phosphorylated form of NF‐κB hindered the reprobing of total NF‐κB. Phosphorylated NF‐κB was normalized to the loading control, with total NF‐κB assessed on a separate gel. (G) qPCR analysis of (i) TNF‐α, (ii) CXCL10, (iii) IL‐23, (iv) CXCL11 transcripts normalized to β‐2M in 4T1 cells 3 h post PEMF exposure ( n = 3 with 3 technical replicates each). (H) Representative images of invading untreated RAW264.7 macrophages in (i) 0 mT or (ii) 3 mT conditioned media (CM); (iii) 0 mT or (iv) 3 mT CM on DMXAA‐treated macrophages; (v) 0 mT or (vi) 3 mT CM on 100 ng/mL IL‐4‐treated macrophages. Scale bar = 100 μm. (I) Bar chart showing the corresponding quantifications. 4T1 cells were conditioned for 24 h to generate CM. Macrophages were incubated in CM for 48 h prior to imaging and analysis ( n = 4 with 2 technical replicates each). All samples were normalized to their time/drug‐matched 0 mT control. Data represent the mean ± the standard error of the mean (SEM). Statistical analysis was performed using Student's unpaired t ‐test (G), one‐way (F) or two‐way (B, D, I) ANOVA, followed by Šidák's multiple comparison post hoc test. Statistical significance is indicated by *, p ≤ 0.05; **, p ≤ 0.01, and *** p ≤ 0.001.

Article Snippet: AP‐18 (10 μM), RN‐9893 (1 μM), 5,6‐dimethylxanthenone‐4‐acetic acid (DMXAA) (20 μg/mL), C176 (2 μM), and Amlexanox (100 μM) were purchased from MedChemExpress.

Techniques: Western Blot, Incubation, Control, Binding Assay, Imaging, Comparison

Journal: Smart Medicine

Article Title: Magnetic Reprogramming of Macrophages Stimulates Phagocytosis of Breast Cancer Cells via a TRPC1‐STING Inflammatory Axis

doi: 10.1002/smmd.70038

Figure Lengend Snippet: PEMF enhances macrophage‐mediated cancer cell clearance via STING‐ and TRPC1‐dependent phagocytosis. Viability of 4T1 cells co‐cultured with RAW264.7 macrophages 24 h post‐PEMF, assessed by (A) CyQuant (DNA content) and (B) MTT (metabolic activity) ( n = 4 with 4 technical replicates each). (C) Representative images of colony‐forming assay on day 7 and (D) quantification of (i) total area and (ii) number of colonies ( n = 3 with 3 technical replicates each). (E) Flow cytometry analysis of co‐cultures represented as (E) dot plots and (F) cell proportions shown as (i) stacked or (ii) separated bars. ( n = 3 with 2 technical replicates each). (G) Representative scatter dot plots and the corresponding quantification (H) of phagocytosis of 4T1 by RAW264.7 macrophages assayed by flow cytometry, showing (i) no CSFE control, (ii) DMXAA (20 μg/mL), (iii–iv) 0 versus 3 mT PEMF; (iii); (v–vi) ± PEMF with STING inhibitor (2 μM C176); (vii–viii) ± PEMF with streptomycin. ( n = 4 with 3 technical replicates each). (I) 4T1 viability in co‐cultures with phagocytosis inhibitor (1 μM Cytochalasin B), TRPC1 inhibitors (5 μM SKF‐96365 or streptomycin), STING inhibitor (2 μM C176), IRF3 inhibitor (0.1 μM BX795) or cGAS‐KO macrophages. ( n = 3 with 8 technical replicates each). (J) (i) Representative western blots and (ii) quantification of CD47 expression on 4T1 cells treated with PEMF‐conditioned media from RAW264.7 macrophages. Data represent the mean ± the standard error of the mean (SEM). Statistical analysis was performed using Student's unpaired t ‐test (D, J) or two‐way (A, B, F, H, I) ANOVA, followed by Šidák's multiple comparison post hoc test with *, p ≤ 0.05; **, p ≤ 0.01 and ***, p ≤ 0.001.

Article Snippet: AP‐18 (10 μM), RN‐9893 (1 μM), 5,6‐dimethylxanthenone‐4‐acetic acid (DMXAA) (20 μg/mL), C176 (2 μM), and Amlexanox (100 μM) were purchased from MedChemExpress.

Techniques: Cell Culture, CyQUANT Assay, Activity Assay, Flow Cytometry, Control, Western Blot, Expressing, Comparison

Journal: Smart Medicine

Article Title: Magnetic Reprogramming of Macrophages Stimulates Phagocytosis of Breast Cancer Cells via a TRPC1‐STING Inflammatory Axis

doi: 10.1002/smmd.70038

Figure Lengend Snippet: PEMF exposure drives tumor cell suppression and enhances TRPC1‐STING‐dependent phagocytosis in spheroids. (A, B) Representative flow cytometry dot plots analyzing cell populations within 4T1‐RAW264.7 co‐culture spheroids at 24 and 48 h post PEMF exposure, respectively. (C, D) Quantified cellular composition of spheroids corresponding to the plots in A and B, respectively. Data are presented as (i) separated or (ii) stacked bars showing the proportions of macrophages (CD45 + ) and cancer cells (CD45 − ). Data are from n = 4‐5 independent experiments, as indicated by dots in the bar charts, with 3 technical replicates each (individual spheroids). (E) Flow cytometry analysis of macrophage phagocytic activity. Representative scatter plots show the percentage of phagocytosing macrophages (CD45 + CFSE + ) under the following conditions: (i)–(ii) ± PEMF exposure, (iii)–(iv) ± PEMF exposure with STING inhibitor C176 (2 μM), (v)–(vi) ± PEMF exposure in the presence of TRPC1 inhibitor streptomycin ( n = 3). (F) Quantification of the phagocytosis percentage, corresponding to the conditions in panel E. (G) Median fluorescence intensity (MFI) of CFSE within the CD45 + macrophage population, indicating phagocytic load. Dots represent individual biological replicates. Values represent mean ± SEM. Statistical analysis was performed using two‐way ANOVA, followed by Šidák's multiple comparison post hoc test with *, p ≤ 0.05; **, p ≤ 0.01, *** and p ≤ 0.001.

Article Snippet: AP‐18 (10 μM), RN‐9893 (1 μM), 5,6‐dimethylxanthenone‐4‐acetic acid (DMXAA) (20 μg/mL), C176 (2 μM), and Amlexanox (100 μM) were purchased from MedChemExpress.

Techniques: Flow Cytometry, Co-Culture Assay, Activity Assay, Fluorescence, Comparison