Journal: Cancer Cell International

Article Title: EGCG inhibits the inflammation and senescence inducing properties of MDA-MB-231 triple-negative breast cancer (TNBC) cells-derived extracellular vesicles in human adipose-derived mesenchymal stem cells

doi: 10.1186/s12935-023-03087-2

Figure Lengend Snippet: Characterization of the EVs isolated from the MDA-MB-231 cells conditioned media . Serum-starved triple-negative breast cancer-derived MDA-MB-231 cells were cultured for 48 h in the absence or presence of 30 µM EGCG. Conditioned media was next collected, concentrated, and extracellular vesicles (EVs) isolated as described in the Methods section. Dynamic light scattering particle size analysis of the A EVs and B EGCG-EVs distribution of the particles by number (upper panels) and by intensity (lower panels) of the refracted light. A representative experiment out of four is presented. C Immunoblotting of the exosomes enriched proteins CD9, CD63 and CD81, and of the negative marker BIP in MDA-MB-231 cell lysate and EVs lysate. D Gating strategy of the flow cytometry experiment performed to assess MemGlow-488 labelled EVs interaction with hADMSC. Merged histogram was obtained by the measurement by flow cytometry of the untreated cells (black lines) and the cells incubated with stained-EVs (red lines). A representative experiment out of two is presented. E Representative microscopy images of hADMSC incubated for two hours with the MDA-MB-231 cells-derived EVs labelled with MemGlow-488. A representative experiment out of three is presented (scale bar is 20 µm). F Relative cell migration rate of hADMSC treated with EVs (closed circle) or basal media (BM, open circle) in response to basal media supplemented with 1% FBS. Migration experiments were performed three times in quadruplicate. Diameter in nanometers (d.nm), polydispersity index (Pdl), intensity weighted mean hydrodynamic size of the particles (Z-Average). Statistically significant differences were determined by the non-parametric comparison test Mann–Whitney, * p < 0.05

Article Snippet: The human adipose-derived mesenchymal stem cells (hADMSC) and TNBC-derived cell line MDA-MB-231 were purchased from American Type Culture Collection (ATCC, Manassas, VA). hADMSC were grown in Mesenchymal Stem Cell Basal Medium (ATCC, PCS-500-030) and supplemented with Mesenchymal Stem Cell Growth Kit Low Serum (ATCC, PCS-500-040).

Techniques: Isolation, Derivative Assay, Cell Culture, Particle Size Analysis, Western Blot, Marker, Flow Cytometry, Incubation, Staining, Microscopy, Migration, Comparison, MANN-WHITNEY

Journal: Cancer Cell International

Article Title: EGCG inhibits the inflammation and senescence inducing properties of MDA-MB-231 triple-negative breast cancer (TNBC) cells-derived extracellular vesicles in human adipose-derived mesenchymal stem cells

doi: 10.1186/s12935-023-03087-2

Figure Lengend Snippet: Signalling cascades triggered by the EVs . The hADMSC were incubated for one hour in basal media (BM), EVs, or EGCG-EVs using a Cell:EVs ratio of 1:0,5 (#:#). Cells were next lysed as described in the Methods section for Western blotting analysis. A phospho-kinase array was used to detect pathways activation states. A Immunoblotting results (1, p38a; 2, STAT5a/b; 3, p53; 4, Chk-2; 5, c-Jun; 6, Akt 1/2/3; 7, GSK-3b), and B Densitometric analysis of the highlighted immunoreactive spots performed using the ImageJ software. C Validation of the phosphorylated and total states of GSK-3β (Ser9) and AKT (Ser473) by immunoblotting. A representative experiment out of two is presented. D Ratios of the phosphorylated/total forms of AKT and GSK-3β resulted from the densitometric analysis performed with ImageJ. Mitogen-activated protein kinases (p38); signal transducer and activator of transcription 5A/B (STAT5a/b); Checkpoint kinase-2 (Chk-2); c-Jun N-terminal kinases JNK (c-Jun); protein kinase B signaling pathway (AKT); glycogen synthase kinase-3 (GSK-3); tumor protein 53 (p53)

Article Snippet: The human adipose-derived mesenchymal stem cells (hADMSC) and TNBC-derived cell line MDA-MB-231 were purchased from American Type Culture Collection (ATCC, Manassas, VA). hADMSC were grown in Mesenchymal Stem Cell Basal Medium (ATCC, PCS-500-030) and supplemented with Mesenchymal Stem Cell Growth Kit Low Serum (ATCC, PCS-500-040).

Techniques: Incubation, Western Blot, Activation Assay, Software, Biomarker Discovery

Journal: Cancer Cell International

Article Title: EGCG inhibits the inflammation and senescence inducing properties of MDA-MB-231 triple-negative breast cancer (TNBC) cells-derived extracellular vesicles in human adipose-derived mesenchymal stem cells

doi: 10.1186/s12935-023-03087-2

Figure Lengend Snippet: Induction of a pro-inflammatory molecular signature by the MDA-MB-231 cells-derived EVs . The hADMSC were incubated for 24 h in Basal Media (BM, Control), EVs (white bars) or EGCG-EVs (black bars) with a Cell: EVs ratio of 1:0,5. Next, total RNA was isolated, and cDNA was synthesized. Gene expression levels were determined by qPCR using a Human Inflammatory Cytokine and Receptors RT2-Profiler gene array kit. Densitometric analysis was performed using the ImageJ software. A The fold change (FC) expression of genes related to the cancer-associated adipocyte (CAA) phenotype. Validation of the arrays results for CAA genes was performed in two independent experiments. B Immunoblotting of interleukin-6 (IL-6) and tubulin (10 µg protein/well). Immunoblotting is representative of three experiments. C FC of selected genes from the array to highlight the modulatory effect of the EGCG-EVs

Article Snippet: The human adipose-derived mesenchymal stem cells (hADMSC) and TNBC-derived cell line MDA-MB-231 were purchased from American Type Culture Collection (ATCC, Manassas, VA). hADMSC were grown in Mesenchymal Stem Cell Basal Medium (ATCC, PCS-500-030) and supplemented with Mesenchymal Stem Cell Growth Kit Low Serum (ATCC, PCS-500-040).

Techniques: Derivative Assay, Incubation, Control, Isolation, Synthesized, Gene Expression, Software, Expressing, Biomarker Discovery, Western Blot

Journal: Cancer Cell International

Article Title: EGCG inhibits the inflammation and senescence inducing properties of MDA-MB-231 triple-negative breast cancer (TNBC) cells-derived extracellular vesicles in human adipose-derived mesenchymal stem cells

doi: 10.1186/s12935-023-03087-2

Figure Lengend Snippet: EGCG-EVs rescue hADMSC from serum-starvation-induced senescence . hADMSC were incubated for 24 h in complete media (CM), serum-deprived basal media (BM), EVs or EGCG-EVs at a ratio Cell:EVs of 1:0.5. hADMSC were collected for protein and total RNA as described in the Methods section. A Immunoblotting detection of the senescence biomarker p21 and of the loading control tubulin from control hADMSC lysates, treated with CM, BM, or the respective EVs. Immunoblotting is representative of three independent experiments. B Confocal microscopy of hADMSC treated for 48 h at a Cell: EVs ratio of 1:2. The nucleus was stained with DAPI (red), and the expression of the senescence-associated β-galactosidase (β-gal) marker is coloured in green. One out of three experiments is presented. C Histograms showing the percent of positive β-gal cells obtained upon 48 h of treatment. D Gene expression of other senescence markers modulated in hADMSC by EGCG-EVs (black bars) compared with the expression level of the genes in cells incubated with EVs (white bars), using as cut-off a log2 FC ≥ 2 and quantified by qPCR using the Human Senescence RT2-Profiler gene array kit. The percent of positive β-gal cells/field was calculated using the following equation: (number of positive cells /total of cells)*100. The Kruskall-Wallis test determined statistically significant differences, showing a * p < 0.05

Article Snippet: The human adipose-derived mesenchymal stem cells (hADMSC) and TNBC-derived cell line MDA-MB-231 were purchased from American Type Culture Collection (ATCC, Manassas, VA). hADMSC were grown in Mesenchymal Stem Cell Basal Medium (ATCC, PCS-500-030) and supplemented with Mesenchymal Stem Cell Growth Kit Low Serum (ATCC, PCS-500-040).

Techniques: Incubation, Western Blot, Biomarker Discovery, Control, Confocal Microscopy, Staining, Expressing, Marker, Gene Expression

Journal: Cancer Cell International

Article Title: EGCG inhibits the inflammation and senescence inducing properties of MDA-MB-231 triple-negative breast cancer (TNBC) cells-derived extracellular vesicles in human adipose-derived mesenchymal stem cells

doi: 10.1186/s12935-023-03087-2

Figure Lengend Snippet: EGCG reduces the mitochondrial content within the EVs . Serum-starved MDA-MB-231 cells were cultured for 48 h in the presence or absence of 30 µM EGCG. EVs were isolated, stained with anti-CD44-FITC and MitoTracker Deep Red (MTR), and analyzed by flow cytometry. Four independent experiments were spaced in time from cell passage 3 to 8. The dots represent the mean of the counting, and results from the same experimental day were connected with a line in graphs A-C. Paired t -test was performed; ** P < 0.01. A The total number of CD44 + /MTR − microparticles (MPs) detected in EVs or EGCG-EVs. B Quantification of the mitochondria-containing particles (CD44 + /MTR + , mitoMPs) in the EVs or EGCG-EVs. Four independent experiments were performed. C Citrate synthase activity was measured in particles isolated from the conditioned media as described in the Methods section. D Dot plot resulting from the flow cytometry analysis detecting the presence of mitochondria delivered by the EVs in hADMSC after incubation with basal media (BM, negative control), mitoTracker-labelled EVs (MTR-EVs), or mitoTracker-labelled ECGC-EVs (MTR-EGCG-EVs). E Mean of the fluorescence intensity of the EVs or EGCG-EVs -delivered mitochondria within hADMSC. F The percent of hADMSC positive for the presence of mitochondria delivered by the EVs or EGCG-EVs

Article Snippet: The human adipose-derived mesenchymal stem cells (hADMSC) and TNBC-derived cell line MDA-MB-231 were purchased from American Type Culture Collection (ATCC, Manassas, VA). hADMSC were grown in Mesenchymal Stem Cell Basal Medium (ATCC, PCS-500-030) and supplemented with Mesenchymal Stem Cell Growth Kit Low Serum (ATCC, PCS-500-040).

Techniques: Cell Culture, Isolation, Staining, Flow Cytometry, Activity Assay, Incubation, Negative Control, Fluorescence