Journal: Nature Communications

Article Title: Apoptotic tumor cell-derived microRNA-375 uses CD36 to alter the tumor-associated macrophage phenotype

doi: 10.1038/s41467-019-08989-2

Figure Lengend Snippet: MΦ uptake of miR-375 as a non-exosome entity. a , b Primary human MΦ were cocultured with MCF-7 cells for 24 h. a One hour before and during the coculture period, MΦ were treated either with vehicle, cytochalasin B, nocodazole, and b carbenoxolone. MiR-375 abundance was quantified via qPCR and normalized to untreated MΦ or untreated coculture MΦ, respectively ( n ≥ 3). c MiR-375 was measured by qPCR in the supernatants of MΦ, viable MCF-7 cells (MCF-7), STS-treated apoptotic MCF-7 cells (ap MCF-7), media, and normalized to untreated MΦ. Synthetic cel-miR-39a was used as spike-in control ( n ≥ 2). d VCM and ACM of ER+ (EFM-192A, MCF-7, T47D) and ER− (MDA-MB-468, MDA-MB-231, SKBR3, HCC1937) breast carcinoma cells, mammary epithelial cells (MCF-10A), and primary mammary epithelial cells (HMEC) were analyzed for the abundance of miR-375 ( n = 3). e MiR-375 level was measured by qPCR in MΦ cocultured with STS-treated apoptotic MCF-7 cells for 4 h. MCF-7 cells were removed from cocultures and MΦ were further cultivated for 20 h (24 h time point). Data are normalized to control MΦ ( n = 5). f MΦ were treated with STS as a control, or 1:1 diluted supernatants of viable (VCM) or apoptotic (ACM) MCF-7 cells for 30 min. Cells were washed and further cultured in MΦ media for 4 and 24 h, and miR-375 abundance was measured and normalized to untreated MΦ ( n ≥ 5). g ACM was incubated with control or 50 µg/mL RNase A at 37 °C for indicated time. Before RNA isolation, cel-miR-39a was added as a normalization control. MiR-375 abundance was quantified by qPCR and normalized to control ACM ( n ≥ 3). h MΦ were incubated for 30 min with either MCF-7 control ACM or RNase A-treated ACM. Cells were washed and cultured for another 24 h in MΦ media. MiR-375 level was determined and normalized to untreated MΦ ( n = 5). Data of a – h are mean ± SEM and p -values were calculated using one-sample t -test. * p < 0.05, ** p < 0.01, *** p < 0.001; n.s., not significant

Article Snippet: Primer for cel-miR-39a was from Qiagen.

Techniques: Cell Culture, Incubation, Isolation

Journal: Nature Communications

Article Title: Apoptotic tumor cell-derived microRNA-375 uses CD36 to alter the tumor-associated macrophage phenotype

doi: 10.1038/s41467-019-08989-2

Figure Lengend Snippet: MCF-7 cell-derived miR-375 is taken up by MΦ via CD36. a MCF-7 cell ACM was prepared with (+ FCS) or without (− FCS) FCS, and miR-375 and miR-183-5p abundance was measured by qPCR. Synthetic cel-miR-39a was used as spike-in control for RNA purification efficiency and normalization control ( n = 4). b LDL and HDL fractions were separated from ACM (with or without FCS in the media) and analyzed for the abundance of miR-375 and miR-183-5p by qPCR ( n = 3). c , d MΦ were pre-incubated with IgG control or anti-CD36 blocking mAb for 1 h and during the whole experiment. c MΦ were treated with ACM for 30 min. Cells were washed and further cultured in MΦ media for 24 h. MiR-375 abundance was measured by qPCR and normalized to untreated control MΦ ( n = 7). d MΦ were cocultured with MCF-7 cells for 24 h and miR-375 abundance was measured via qPCR, and normalized to untreated control MΦ ( n = 4). e MΦ were treated with MCF-7 ACM alone or together with a CD36-blocking peptide for 30 min. The peptide was added 1 h before ACM treatment. Afterwards, cells were washed and further cultured in MΦ media alone or together with the peptide for 24 h. miR-375 level was measured by qPCR and normalized to untreated control MΦ ( n = 8). f , g MΦ were transfected with control (nonspecific siRNA) or CD36 siRNA for 24 h and cocultured with MCF-7 cells for another 24 h. f CD36 mRNA expression and g miR-375 levels were measured by qPCR ( n = 6). Data of a – g are mean ± SEM and p -values were calculated using two-tailed Student’s t -test ( a , b , f , g ) and one-sample t -test ( c – e ); * p < 0.05, ** p < 0.01, n.s., not significant

Article Snippet: Primer for cel-miR-39a was from Qiagen.

Techniques: Derivative Assay, Purification, Incubation, Blocking Assay, Cell Culture, Transfection, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Apoptotic tumor cell-derived microRNA-375 uses CD36 to alter the tumor-associated macrophage phenotype

doi: 10.1038/s41467-019-08989-2

Figure Lengend Snippet: PXN and TNS3 are direct targets of miR-375 in human MΦ. a , c MΦ were transfected with synthetic miR-375 mimic or cel-miR-39a control (scramble) for 72 h. a mRNA and protein expression of PXN and TNS3 was determined. Data are normalized to transfection controls for mRNA and nucleolin for protein ( n ≥ 3). b Relative PXN and TNS3 mRNA and protein expression in ACM-treated MΦ were normalized to control MΦ or nucleolin, respectively ( n ≥ 4). c mRNA stability of PXN and TNS3 was measured. MΦ were treated with actinomycin D for 0–8 h. PXN and TNS3 mRNA contents at the time of adding actinomycin D was set to 100%. PXN and TNS3 mRNA expression in control-transfected MΦ (gray circle) and miR-375-overexpressing MΦ (black square) was measured at indicated time by qPCR ( n ≥ 5). mRNA half-life ( t 1/2 ) was calculated by exponential regression curve. d MΦ were transfected with 2 µg PXN or TNS3 3′-UTR reporter plasmids or empty control vector with or without synthetic miR-375 mimic for 48 h. Binding of miR-375 to its target genes was analyzed as the ratio of renilla luciferase activity to firefly luciferase activity ( n = 4). e – g Primary human MΦ were transfected with control or PXN and TNS3 siRNA for 24 h. After 24 h, e MΦ were collected and PXN and TNS3 expression was analyzed by qPCR or f , g treated with MCF-7 control or decoy ACM for 30 min. Cells were washed and fresh MΦ media was added for 24 h. Scratches were generated with a small pipette tip in a marked area. Pictures were taken at 0 and 24 h, and the cell-free area within the scratch was measured for percental gap closure normalized to untreated MΦ control (MΦ control = 0%) using ImageJ software ( n ≥ 5). Data of a – f are mean ± SEM and p -values were calculated using one-sample t -test ( a , b , d , e ) and two-way ANOVA with Bonferroni’s correction ( f ). * p < 0.05, ** p < 0.01, *** p < 0.001; n.s., not significant

Article Snippet: Primer for cel-miR-39a was from Qiagen.

Techniques: Transfection, Expressing, Plasmid Preparation, Binding Assay, Luciferase, Activity Assay, Generated, Transferring, Software

Journal: Blood

Article Title: Serum free light chains, not urine specimens, should be used to evaluate response in light-chain multiple myeloma

doi: 10.1182/blood-2016-07-726778

Figure Lengend Snippet: Survival outcomes according to urine FLC and sFLC characteristics at the end of induction therapy. PFS for patients with (A) negative vs positive UPEP (median PFS, 47 and 36 months, respectively), (B) normal vs elevated iFLC (median PFS, not reached and 36 months, respectively), (C) negative vs positive uIFE (median PFS, 47 and 36 months, respectively), and (D) normal vs abnormal κ:λ sFLC ratio (median PFS, not reached and 34 months, respectively). OS for patients with (E) negative vs positive uIFE (median OS not reached for both) and (F) normal vs abnormal κ:λ sFLC ratio (median OS not reached for both). P values calculated by log-rank test. Number of patients (events) for each arm is indicated. Comparisons include patients with matched urine and serum data.

Article Snippet: Urine protein electrophoresis (UPEP) and urine immunofixation electrophoresis (uIFE; Sebia) were performed prospectively throughout the trial, using standard laboratory procedures on neat samples. sFLC concentrations were measured using κ sFLC and λ sFLC Freelite assays (The Binding Site Group Ltd) on a BN II nephelometer (Siemens).

Techniques:

Journal: Blood

Article Title: Serum free light chains, not urine specimens, should be used to evaluate response in light-chain multiple myeloma

doi: 10.1182/blood-2016-07-726778

Figure Lengend Snippet: Abnormal sFLC measurements stratify patients with normal urine results after induction. PFS according to (A) normal vs elevated iFLC in patients with a negative UPEP result (median PFS, not reached and 34 months, respectively), (B) negative vs positive UPEP in patients with elevated iFLC (median PFS, 34 and 36 months, respectively), and (C) normal vs abnormal κ:λ sFLC ratio in patients with negative uIFE (median PFS, not reached and 31 months, respectively). (D) OS for patients with normal vs abnormal κ:λ sFLC ratio in patients with negative uIFE (median OS not reached for both). P values calculated by log-rank test. Number of patients (events) for each arm is indicated. Comparisons include patients with matched urine and serum data.

Article Snippet: Urine protein electrophoresis (UPEP) and urine immunofixation electrophoresis (uIFE; Sebia) were performed prospectively throughout the trial, using standard laboratory procedures on neat samples. sFLC concentrations were measured using κ sFLC and λ sFLC Freelite assays (The Binding Site Group Ltd) on a BN II nephelometer (Siemens).

Techniques: