Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: Aged fibroblasts secrete high levels of IGFBP2. A, Re-analysis of proteomics on CM from young and aged dermal fibroblasts, published in Kaur and colleagues , showing differentially expressed proteins between the two groups, in which red denotes increased expression in aged as compared with young, and blue denotes decreased expression. B, IGFBP2 ELISA analysis in young and aged human dermal fibroblast CM ( P = 0.0055). C, IGFBP2 staining in human melanoma skin reconstructs with young or aged donor-derived dermal fibroblasts. D, IGFBP2 staining in primary tumor tissue from young and aged C57BL6 mice. E, RPPA analysis of young and aged YUMM1.7 mouse tumor lysate for IGFBP2 expression ( P = 0.002). F, Pathway analysis of RPPA analysis of young and aged YUMM1.7 mouse tumor lysate. G, P-AKT (Ser473) staining of tumors in young and aged mice. H, Oil Red O staining of tumors in young and aged mice.

Article Snippet: Briefly, 500 μL of growth medium (20% FBS) was added to the bottom of each well, and a total of 2.5 × 10 4 cells resuspended in 250 μL of young conditioned media (CM) in the presence or absence of rIGFBP2 (150 ng/mL; 674-B2-025 from R&D Systems) or aged CM in the presence or absence of 5 mg/mL neutralizing IGFBP2 antibody (AF674 from R&D Systems) were seeded on top.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Derivative Assay

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: IGFBP2 induces fatty acid synthesis in melanoma cells. A, Western blot analysis of Igfbp2, phospho-Akt, total Akt and Hsp90 (loading control) from young and aged tumor lysate. Quantification of immunoblotting of Igfbp2 and phospho-Akt relative to Hsp90 loading control. B, Correlation analysis of IGFBP2 protein expression and p-AKT S473 expression from RPPA of melanoma PDX samples. C, Correlation analysis of IGFBP2 protein expression and p-AKT T308 expression from RPPA of melanoma PDX samples. D, Western blot analysis of melanoma cells (1205Lu) treated with recombinant IGFBP2 (100 ng/mL) at different times. Cells were probed for IGFBP2, phospho-AKT, total AKT, and HSP90 (loading control). E, Western blot analysis of IGFBP2 knockdown in aged fibroblasts. F, BODIPY (505/515) staining of human melanoma cells (1205Lu) cultured with aged fibroblast CM from fibroblasts transduced with empty vector (PLKO.1) or shRNA constructs (shIGFBP2). Quantification of BODIPY. G, BODIPY (505/515) staining of melanoma cells treated with CM from young fibroblasts treated with recombinant IGFBP2 (50 and 100 ng/mL). Quantification of BODIPY. H, RT-PCR of IGFBP2 expression in melanoma cells after treatment with young or aged CM. I, Western blot analysis of IGFBP2, P-AKT, total AKT, and HSP90 in melanoma cells transfected with either an empty vector control (CTRL) or IGFBP2 (IGFBP2 OE). J, BODIPY (493/503) staining of melanoma cells after transfection with either control or IGFBP2 constructs with quantification. GraphPad Prism 8 was used for plotting graphs and statistical analysis. Unpaired t test was performed.

Article Snippet: Briefly, 500 μL of growth medium (20% FBS) was added to the bottom of each well, and a total of 2.5 × 10 4 cells resuspended in 250 μL of young conditioned media (CM) in the presence or absence of rIGFBP2 (150 ng/mL; 674-B2-025 from R&D Systems) or aged CM in the presence or absence of 5 mg/mL neutralizing IGFBP2 antibody (AF674 from R&D Systems) were seeded on top.

Techniques: Western Blot, Control, Expressing, Recombinant, Knockdown, Staining, Cell Culture, Transduction, Plasmid Preparation, shRNA, Construct, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: IGFBP2 increases melanoma cell migration and invasion. A, Wound healing assay of human melanoma cells (1205Lu, WM164) cultured with young or aged CM in the presence or absence of a neutralizing IGFBP2 antibody (5 mg/mL) or recombinant IGFBP2 (150 ng/mL). B, Matrigel invasion assay of melanoma cells (1205Lu, WM164) cultured with young and aged CM in the presence or absence of a neutralizing IGFBP2 antibody or recombinant IGFBP2. C, Melanoma cells (1205Lu) grown in 3D spheroids cultured with aged CM and young CM in the presence or absence of recombinant IGFBP2 (150 ng/mL) for 48 hours. GraphPad Prism 8 was used for plotting graphs and statistical analysis. Unpaired t test was performed.

Article Snippet: Briefly, 500 μL of growth medium (20% FBS) was added to the bottom of each well, and a total of 2.5 × 10 4 cells resuspended in 250 μL of young conditioned media (CM) in the presence or absence of rIGFBP2 (150 ng/mL; 674-B2-025 from R&D Systems) or aged CM in the presence or absence of 5 mg/mL neutralizing IGFBP2 antibody (AF674 from R&D Systems) were seeded on top.

Techniques: Migration, Wound Healing Assay, Cell Culture, Recombinant, Invasion Assay

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: Igfbp2 increases melanoma tumor growth and metastasis in vivo . A, mCherry-tagged YUMM1.7 murine melanoma cells were grown in young (8 weeks old) C57BL6 mice. Tumor growth of young mice after i.p. treatment with 500 ng recombinant Igfbp2 or PBS (5 mice/group, treated i.p., 2 times a week, after tumors were palpable). B, IHC analysis of Igfbp2 in tumors from young mice treated with PBS or recombinant Igfbp2. C, Western blot analysis of tumor lysate from young mice treated with PBS and recombinant Igfbp2. Densitometry of phospho-AKT relative to HSP90 loading control. D, Tumor growth curve of YUMM1.7 melanoma cells subdermally injected in old (52 weeks old) mice treated i.p. with Igfbp2-neutralizing antibody (at a concentration of 1 mg/kg every day, n = 5) vs. an IgG control ( n = 5). E, IHC analysis of Igfbp2 staining in aged mice treated with either an IgG control or neutralizing Igfbp2 antibody. F, Protein expression analysis was performed on tumor lysate from aged mice treated with IgG or neutralizing Igfbp2 antibody. Quantification analysis of phospho-Akt and Fasn immunoblotting relative to Hsp90 loading control. G, Analysis of mCherry-positive cells in lungs of tumor-bearing aged mice treated with a neutralizing Igfbp2 antibody or IgG control. *, P < 0.05 student t test was used. GraphPad Prism 8 was used for plotting graphs and statistical analysis.

Article Snippet: Briefly, 500 μL of growth medium (20% FBS) was added to the bottom of each well, and a total of 2.5 × 10 4 cells resuspended in 250 μL of young conditioned media (CM) in the presence or absence of rIGFBP2 (150 ng/mL; 674-B2-025 from R&D Systems) or aged CM in the presence or absence of 5 mg/mL neutralizing IGFBP2 antibody (AF674 from R&D Systems) were seeded on top.

Techniques: In Vivo, Recombinant, Western Blot, Control, Injection, Concentration Assay, Staining, Expressing

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: Aged fibroblasts secrete high levels of IGFBP2. A, Re-analysis of proteomics on CM from young and aged dermal fibroblasts, published in Kaur and colleagues , showing differentially expressed proteins between the two groups, in which red denotes increased expression in aged as compared with young, and blue denotes decreased expression. B, IGFBP2 ELISA analysis in young and aged human dermal fibroblast CM ( P = 0.0055). C, IGFBP2 staining in human melanoma skin reconstructs with young or aged donor-derived dermal fibroblasts. D, IGFBP2 staining in primary tumor tissue from young and aged C57BL6 mice. E, RPPA analysis of young and aged YUMM1.7 mouse tumor lysate for IGFBP2 expression ( P = 0.002). F, Pathway analysis of RPPA analysis of young and aged YUMM1.7 mouse tumor lysate. G, P-AKT (Ser473) staining of tumors in young and aged mice. H, Oil Red O staining of tumors in young and aged mice.

Article Snippet: Tumors were allowed to grow, and treatment with rIgfbp2 (674-B2-025 from R&D Systems), Igfbp2-neutralizing antibody (AF674 from R&D Systems), or IgG control (AB-105-C from R&D Systems) was administered. rIGFBP2 experiments: young mice were injected intraperitoneally with 500 ng of rIgfbp2 in 100 μL of PBS every 2 days until tumors reach a maximum of 2,000 mm 3 .

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Derivative Assay

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: IGFBP2 induces fatty acid synthesis in melanoma cells. A, Western blot analysis of Igfbp2, phospho-Akt, total Akt and Hsp90 (loading control) from young and aged tumor lysate. Quantification of immunoblotting of Igfbp2 and phospho-Akt relative to Hsp90 loading control. B, Correlation analysis of IGFBP2 protein expression and p-AKT S473 expression from RPPA of melanoma PDX samples. C, Correlation analysis of IGFBP2 protein expression and p-AKT T308 expression from RPPA of melanoma PDX samples. D, Western blot analysis of melanoma cells (1205Lu) treated with recombinant IGFBP2 (100 ng/mL) at different times. Cells were probed for IGFBP2, phospho-AKT, total AKT, and HSP90 (loading control). E, Western blot analysis of IGFBP2 knockdown in aged fibroblasts. F, BODIPY (505/515) staining of human melanoma cells (1205Lu) cultured with aged fibroblast CM from fibroblasts transduced with empty vector (PLKO.1) or shRNA constructs (shIGFBP2). Quantification of BODIPY. G, BODIPY (505/515) staining of melanoma cells treated with CM from young fibroblasts treated with recombinant IGFBP2 (50 and 100 ng/mL). Quantification of BODIPY. H, RT-PCR of IGFBP2 expression in melanoma cells after treatment with young or aged CM. I, Western blot analysis of IGFBP2, P-AKT, total AKT, and HSP90 in melanoma cells transfected with either an empty vector control (CTRL) or IGFBP2 (IGFBP2 OE). J, BODIPY (493/503) staining of melanoma cells after transfection with either control or IGFBP2 constructs with quantification. GraphPad Prism 8 was used for plotting graphs and statistical analysis. Unpaired t test was performed.

Article Snippet: Tumors were allowed to grow, and treatment with rIgfbp2 (674-B2-025 from R&D Systems), Igfbp2-neutralizing antibody (AF674 from R&D Systems), or IgG control (AB-105-C from R&D Systems) was administered. rIGFBP2 experiments: young mice were injected intraperitoneally with 500 ng of rIgfbp2 in 100 μL of PBS every 2 days until tumors reach a maximum of 2,000 mm 3 .

Techniques: Western Blot, Control, Expressing, Recombinant, Knockdown, Staining, Cell Culture, Transduction, Plasmid Preparation, shRNA, Construct, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: IGFBP2 increases melanoma cell migration and invasion. A, Wound healing assay of human melanoma cells (1205Lu, WM164) cultured with young or aged CM in the presence or absence of a neutralizing IGFBP2 antibody (5 mg/mL) or recombinant IGFBP2 (150 ng/mL). B, Matrigel invasion assay of melanoma cells (1205Lu, WM164) cultured with young and aged CM in the presence or absence of a neutralizing IGFBP2 antibody or recombinant IGFBP2. C, Melanoma cells (1205Lu) grown in 3D spheroids cultured with aged CM and young CM in the presence or absence of recombinant IGFBP2 (150 ng/mL) for 48 hours. GraphPad Prism 8 was used for plotting graphs and statistical analysis. Unpaired t test was performed.

Article Snippet: Tumors were allowed to grow, and treatment with rIgfbp2 (674-B2-025 from R&D Systems), Igfbp2-neutralizing antibody (AF674 from R&D Systems), or IgG control (AB-105-C from R&D Systems) was administered. rIGFBP2 experiments: young mice were injected intraperitoneally with 500 ng of rIgfbp2 in 100 μL of PBS every 2 days until tumors reach a maximum of 2,000 mm 3 .

Techniques: Migration, Wound Healing Assay, Cell Culture, Recombinant, Invasion Assay

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: Igfbp2 increases melanoma tumor growth and metastasis in vivo . A, mCherry-tagged YUMM1.7 murine melanoma cells were grown in young (8 weeks old) C57BL6 mice. Tumor growth of young mice after i.p. treatment with 500 ng recombinant Igfbp2 or PBS (5 mice/group, treated i.p., 2 times a week, after tumors were palpable). B, IHC analysis of Igfbp2 in tumors from young mice treated with PBS or recombinant Igfbp2. C, Western blot analysis of tumor lysate from young mice treated with PBS and recombinant Igfbp2. Densitometry of phospho-AKT relative to HSP90 loading control. D, Tumor growth curve of YUMM1.7 melanoma cells subdermally injected in old (52 weeks old) mice treated i.p. with Igfbp2-neutralizing antibody (at a concentration of 1 mg/kg every day, n = 5) vs. an IgG control ( n = 5). E, IHC analysis of Igfbp2 staining in aged mice treated with either an IgG control or neutralizing Igfbp2 antibody. F, Protein expression analysis was performed on tumor lysate from aged mice treated with IgG or neutralizing Igfbp2 antibody. Quantification analysis of phospho-Akt and Fasn immunoblotting relative to Hsp90 loading control. G, Analysis of mCherry-positive cells in lungs of tumor-bearing aged mice treated with a neutralizing Igfbp2 antibody or IgG control. *, P < 0.05 student t test was used. GraphPad Prism 8 was used for plotting graphs and statistical analysis.

Article Snippet: Tumors were allowed to grow, and treatment with rIgfbp2 (674-B2-025 from R&D Systems), Igfbp2-neutralizing antibody (AF674 from R&D Systems), or IgG control (AB-105-C from R&D Systems) was administered. rIGFBP2 experiments: young mice were injected intraperitoneally with 500 ng of rIgfbp2 in 100 μL of PBS every 2 days until tumors reach a maximum of 2,000 mm 3 .

Techniques: In Vivo, Recombinant, Western Blot, Control, Injection, Concentration Assay, Staining, Expressing

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: Aged fibroblasts secrete high levels of IGFBP2. A, Re-analysis of proteomics on CM from young and aged dermal fibroblasts, published in Kaur and colleagues , showing differentially expressed proteins between the two groups, in which red denotes increased expression in aged as compared with young, and blue denotes decreased expression. B, IGFBP2 ELISA analysis in young and aged human dermal fibroblast CM ( P = 0.0055). C, IGFBP2 staining in human melanoma skin reconstructs with young or aged donor-derived dermal fibroblasts. D, IGFBP2 staining in primary tumor tissue from young and aged C57BL6 mice. E, RPPA analysis of young and aged YUMM1.7 mouse tumor lysate for IGFBP2 expression ( P = 0.002). F, Pathway analysis of RPPA analysis of young and aged YUMM1.7 mouse tumor lysate. G, P-AKT (Ser473) staining of tumors in young and aged mice. H, Oil Red O staining of tumors in young and aged mice.

Article Snippet: Melanoma cells were then cultured with young CM in the presence or absence of (150 ng/mL) rIGFBP2 (674-B2-025 from R&D Systems) and aged CM from fibroblasts treated with neutralizing (5 mg/mL) IGFBP2 antibody (AF674 from R&D Systems) for 24 hours.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Derivative Assay

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: IGFBP2 induces fatty acid synthesis in melanoma cells. A, Western blot analysis of Igfbp2, phospho-Akt, total Akt and Hsp90 (loading control) from young and aged tumor lysate. Quantification of immunoblotting of Igfbp2 and phospho-Akt relative to Hsp90 loading control. B, Correlation analysis of IGFBP2 protein expression and p-AKT S473 expression from RPPA of melanoma PDX samples. C, Correlation analysis of IGFBP2 protein expression and p-AKT T308 expression from RPPA of melanoma PDX samples. D, Western blot analysis of melanoma cells (1205Lu) treated with recombinant IGFBP2 (100 ng/mL) at different times. Cells were probed for IGFBP2, phospho-AKT, total AKT, and HSP90 (loading control). E, Western blot analysis of IGFBP2 knockdown in aged fibroblasts. F, BODIPY (505/515) staining of human melanoma cells (1205Lu) cultured with aged fibroblast CM from fibroblasts transduced with empty vector (PLKO.1) or shRNA constructs (shIGFBP2). Quantification of BODIPY. G, BODIPY (505/515) staining of melanoma cells treated with CM from young fibroblasts treated with recombinant IGFBP2 (50 and 100 ng/mL). Quantification of BODIPY. H, RT-PCR of IGFBP2 expression in melanoma cells after treatment with young or aged CM. I, Western blot analysis of IGFBP2, P-AKT, total AKT, and HSP90 in melanoma cells transfected with either an empty vector control (CTRL) or IGFBP2 (IGFBP2 OE). J, BODIPY (493/503) staining of melanoma cells after transfection with either control or IGFBP2 constructs with quantification. GraphPad Prism 8 was used for plotting graphs and statistical analysis. Unpaired t test was performed.

Article Snippet: Melanoma cells were then cultured with young CM in the presence or absence of (150 ng/mL) rIGFBP2 (674-B2-025 from R&D Systems) and aged CM from fibroblasts treated with neutralizing (5 mg/mL) IGFBP2 antibody (AF674 from R&D Systems) for 24 hours.

Techniques: Western Blot, Control, Expressing, Recombinant, Knockdown, Staining, Cell Culture, Transduction, Plasmid Preparation, shRNA, Construct, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: IGFBP2 increases melanoma cell migration and invasion. A, Wound healing assay of human melanoma cells (1205Lu, WM164) cultured with young or aged CM in the presence or absence of a neutralizing IGFBP2 antibody (5 mg/mL) or recombinant IGFBP2 (150 ng/mL). B, Matrigel invasion assay of melanoma cells (1205Lu, WM164) cultured with young and aged CM in the presence or absence of a neutralizing IGFBP2 antibody or recombinant IGFBP2. C, Melanoma cells (1205Lu) grown in 3D spheroids cultured with aged CM and young CM in the presence or absence of recombinant IGFBP2 (150 ng/mL) for 48 hours. GraphPad Prism 8 was used for plotting graphs and statistical analysis. Unpaired t test was performed.

Article Snippet: Melanoma cells were then cultured with young CM in the presence or absence of (150 ng/mL) rIGFBP2 (674-B2-025 from R&D Systems) and aged CM from fibroblasts treated with neutralizing (5 mg/mL) IGFBP2 antibody (AF674 from R&D Systems) for 24 hours.

Techniques: Migration, Wound Healing Assay, Cell Culture, Recombinant, Invasion Assay

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: Igfbp2 increases melanoma tumor growth and metastasis in vivo . A, mCherry-tagged YUMM1.7 murine melanoma cells were grown in young (8 weeks old) C57BL6 mice. Tumor growth of young mice after i.p. treatment with 500 ng recombinant Igfbp2 or PBS (5 mice/group, treated i.p., 2 times a week, after tumors were palpable). B, IHC analysis of Igfbp2 in tumors from young mice treated with PBS or recombinant Igfbp2. C, Western blot analysis of tumor lysate from young mice treated with PBS and recombinant Igfbp2. Densitometry of phospho-AKT relative to HSP90 loading control. D, Tumor growth curve of YUMM1.7 melanoma cells subdermally injected in old (52 weeks old) mice treated i.p. with Igfbp2-neutralizing antibody (at a concentration of 1 mg/kg every day, n = 5) vs. an IgG control ( n = 5). E, IHC analysis of Igfbp2 staining in aged mice treated with either an IgG control or neutralizing Igfbp2 antibody. F, Protein expression analysis was performed on tumor lysate from aged mice treated with IgG or neutralizing Igfbp2 antibody. Quantification analysis of phospho-Akt and Fasn immunoblotting relative to Hsp90 loading control. G, Analysis of mCherry-positive cells in lungs of tumor-bearing aged mice treated with a neutralizing Igfbp2 antibody or IgG control. *, P < 0.05 student t test was used. GraphPad Prism 8 was used for plotting graphs and statistical analysis.

Article Snippet: Melanoma cells were then cultured with young CM in the presence or absence of (150 ng/mL) rIGFBP2 (674-B2-025 from R&D Systems) and aged CM from fibroblasts treated with neutralizing (5 mg/mL) IGFBP2 antibody (AF674 from R&D Systems) for 24 hours.

Techniques: In Vivo, Recombinant, Western Blot, Control, Injection, Concentration Assay, Staining, Expressing