Journal: Cellular & Molecular Biology Letters

Article Title: Melanoma cells induce dedifferentiation and metabolic changes in adipocytes present in the tumor niche

doi: 10.1186/s11658-023-00476-3

Figure Lengend Snippet: Characterization of cancer-associated adipocytes (CAAs). Adipocytes were cocultured with melanoma cells (lines: WM1341D, A375, WM9, and Hs294T) present on Transwell inserts, and then the expression of leptin, adiponectin, resistin, and FABP4 was established in obtained CAAs, as well as nondifferentiated 3T3L1 fibroblasts and control adipocytes, using real-time PCR analysis. For quantification, the level of expression was normalized to the expression of EEF2 and presented as the percent of control (untreated adipocytes). The mean of at least three biological repetitions ± standard deviation (SD) is shown. Asterisks indicate statistically significant differences between control adipocytes and 3T3L1/CAAs. The significance levels were set at p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), or p ≤ 0.0001 (****). FABP4 fatty acid-binding protein 4, EEF2 eukaryotic translation elongation factor 2

Article Snippet: Briefly, 2 days after reaching confluence, cells were treated with a mixture of rosiglitazone (Merck, Darmstadt, Germany), insulin (Sigma Aldrich, Burlington, Massachusetts, USA), dexamethasone (Sigma Aldrich, Burlington, Massachusetts, USA), and 3-isobutyl-1-methylxanthine (IBMX; Merck, Darmstadt, Germany) for 48 h. They were then incubated with insulin for another 48 h. The influence of melanoma cells on adipocytes was assessed using four melanoma cell lines: A375 (CRL-1619) and Hs294T (HTB-140), obtained from the ATCC, and WM1341D (WM1341D-01-0001) and WM9 (WM9-01-0001) cell lines purchased from Rockland Immunochemicals, Inc. (Royersford, Pennsylvania, United States).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Standard Deviation, Binding Assay

Journal: Cellular & Molecular Biology Letters

Article Title: Melanoma cells induce dedifferentiation and metabolic changes in adipocytes present in the tumor niche

doi: 10.1186/s11658-023-00476-3

Figure Lengend Snippet: Features of fibroblasts exhibited by CAAs. A Adipocytes were cocultured with melanoma cells ( lines: WM1341D, A375, WM9 and Hs294T) present on Transwell inserts and then obtained CAAs as well as the non-differentiated 3T3L1 fibroblasts and control adipocytes were seeded on coverslips coated with gelatin-FITC (green). After 16 h of incubation, cells were fixed and stained using phalloidin-Alexa Fluor 568 to visualize F-actin (red). Areas of gelatin degradation are visible as dark holes on a green background. White arrows indicate stress fibers. Scale bar: 25 μm. B Western blot analysis of protein level of vimentin and TGFβ receptor III (TGFβRIII) in cell lysates of 3T3L1 cells; control adipocytes, and adipocytes cultured with melanoma cells growing on Transwell inserts. The signal was normalized to total protein content assessed by Ponceau S staining. The mean of at least three biological repetitions ± SD is shown. Asterisks indicate statistically significant differences between control adipocytes, 3T3L1 fibroblasts and CAAs. The significance levels were set at p ≤ 0.05 (*), p ≤ 0.01 (**), or p ≤ 0.0001 (****).

Article Snippet: Briefly, 2 days after reaching confluence, cells were treated with a mixture of rosiglitazone (Merck, Darmstadt, Germany), insulin (Sigma Aldrich, Burlington, Massachusetts, USA), dexamethasone (Sigma Aldrich, Burlington, Massachusetts, USA), and 3-isobutyl-1-methylxanthine (IBMX; Merck, Darmstadt, Germany) for 48 h. They were then incubated with insulin for another 48 h. The influence of melanoma cells on adipocytes was assessed using four melanoma cell lines: A375 (CRL-1619) and Hs294T (HTB-140), obtained from the ATCC, and WM1341D (WM1341D-01-0001) and WM9 (WM9-01-0001) cell lines purchased from Rockland Immunochemicals, Inc. (Royersford, Pennsylvania, United States).

Techniques: Control, Incubation, Staining, Western Blot, Cell Culture

Journal: Cellular & Molecular Biology Letters

Article Title: Melanoma cells induce dedifferentiation and metabolic changes in adipocytes present in the tumor niche

doi: 10.1186/s11658-023-00476-3

Figure Lengend Snippet: Loss of lipid content in cancer-associated adipocytes. Adipocytes were cocultured with melanoma cells (lines: WM1341D, A375, WM9, and Hs294T) present on Transwell inserts. A After coculture, obtained CAAs, control adipocytes, and 3T3L1 fibroblasts were seeded on coverslips, fixed, and stained with Lipid Spot 488 to visualize lipid droplets. Cell nuclei were stained using Hoechst 33,342 reagent. Scale bar: 25 μm. B To quantify the amount of neutral lipids, cells were fixed and then stained with Oil Red O, and the amount was measured spectrophotometrically. C Expression of PLIN1 (perilipin1) in 3T3L1 cells, control adipocytes, and CAAs established by real-time PCR analysis. For quantification, the samples were normalized against the expression of EEF2. For all experiments, the control constitutes normal adipocytes. In the case of quantitative analyses, the mean of at least three biological repetitions ± SD is shown. Asterisks indicate statistically significant differences between control cells and 3T3L1/CAAs. The significance levels were set at p ≤ 0.01 (**), p ≤ 0.001 (***), or p ≤ 0.0001 (****)

Article Snippet: Briefly, 2 days after reaching confluence, cells were treated with a mixture of rosiglitazone (Merck, Darmstadt, Germany), insulin (Sigma Aldrich, Burlington, Massachusetts, USA), dexamethasone (Sigma Aldrich, Burlington, Massachusetts, USA), and 3-isobutyl-1-methylxanthine (IBMX; Merck, Darmstadt, Germany) for 48 h. They were then incubated with insulin for another 48 h. The influence of melanoma cells on adipocytes was assessed using four melanoma cell lines: A375 (CRL-1619) and Hs294T (HTB-140), obtained from the ATCC, and WM1341D (WM1341D-01-0001) and WM9 (WM9-01-0001) cell lines purchased from Rockland Immunochemicals, Inc. (Royersford, Pennsylvania, United States).

Techniques: Control, Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: Cellular & Molecular Biology Letters

Article Title: Melanoma cells induce dedifferentiation and metabolic changes in adipocytes present in the tumor niche

doi: 10.1186/s11658-023-00476-3

Figure Lengend Snippet: Influence of melanoma cells on lactate secretion ( A ), ions ( B , C ), and glucose transport ( D ) by 3T3L1 cells, control adipocytes, and CAAs. CAAs were obtained from adipocytes cultured with melanoma cells (lines: WM1341D, A375, WM9, and Hs294T) present on Transwell inserts. A The level of secreted lactate was measured using a chemiluminescent reaction in a cell-conditioned medium devoid of serum, in which cells were grown for 24 h. The expression of NHE1 ( B ), MCT1 ( C ), and GLUT1 ( D ) was estimated through real-time PCR analysis. For quantification, the samples were normalized against the expression of EEF2. The mean of at least three independent repetitions ± SD is shown. Asterisks indicate statistically significant differences between control cells and 3T3L1/CAAs. The significance levels were set at p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), or p ≤ 0.0001 (****). NHE1 sodium/hydrogen exchanger 1, MCT1 monocarboxylate transporter 1, GLUT1 glucose transporter 1, EEF2 eukaryotic translation elongation factor 2

Article Snippet: Briefly, 2 days after reaching confluence, cells were treated with a mixture of rosiglitazone (Merck, Darmstadt, Germany), insulin (Sigma Aldrich, Burlington, Massachusetts, USA), dexamethasone (Sigma Aldrich, Burlington, Massachusetts, USA), and 3-isobutyl-1-methylxanthine (IBMX; Merck, Darmstadt, Germany) for 48 h. They were then incubated with insulin for another 48 h. The influence of melanoma cells on adipocytes was assessed using four melanoma cell lines: A375 (CRL-1619) and Hs294T (HTB-140), obtained from the ATCC, and WM1341D (WM1341D-01-0001) and WM9 (WM9-01-0001) cell lines purchased from Rockland Immunochemicals, Inc. (Royersford, Pennsylvania, United States).

Techniques: Control, Cell Culture, Expressing, Real-time Polymerase Chain Reaction

Journal: Cellular & Molecular Biology Letters

Article Title: Melanoma cells induce dedifferentiation and metabolic changes in adipocytes present in the tumor niche

doi: 10.1186/s11658-023-00476-3

Figure Lengend Snippet: Effect of coculture with adipocytes on the proliferation of melanoma cells. Melanoma cells (lines: WM1341D, A375, WM9, and Hs294T) were cocultured with adipocytes present on Transwell inserts for 7 days. Next, melanoma cells were seeded on a 96-well plate, and their proliferation rate was calculated as a ratio of the spectrophotometric signal after 48 h to the spectrophotometric signal at T0. Results are expressed as the mean (fold change) ± SD of three independent experiments. Asterisks above the bars express significance vs. control. The significance levels were set at p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***)

Article Snippet: Briefly, 2 days after reaching confluence, cells were treated with a mixture of rosiglitazone (Merck, Darmstadt, Germany), insulin (Sigma Aldrich, Burlington, Massachusetts, USA), dexamethasone (Sigma Aldrich, Burlington, Massachusetts, USA), and 3-isobutyl-1-methylxanthine (IBMX; Merck, Darmstadt, Germany) for 48 h. They were then incubated with insulin for another 48 h. The influence of melanoma cells on adipocytes was assessed using four melanoma cell lines: A375 (CRL-1619) and Hs294T (HTB-140), obtained from the ATCC, and WM1341D (WM1341D-01-0001) and WM9 (WM9-01-0001) cell lines purchased from Rockland Immunochemicals, Inc. (Royersford, Pennsylvania, United States).

Techniques: Control