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Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin β1/FAK signaling.

Hirata E, Girotti MR, Viros A, Hooper S, Spencer-Dene B, Matsuda M, Larkin J, Marais R, Sahai E - Cancer Cell (2015)

Bottom Line: Intravital imaging of BRAF-mutant melanoma cells containing an ERK/MAPK biosensor reveals how the tumor microenvironment affects response to BRAF inhibition by PLX4720.Fibronectin-rich matrices with 3-12 kPa elastic modulus are sufficient to provide PLX4720 tolerance.We propose that paradoxically activated MAFs provide a "safe haven" for melanoma cells to tolerate BRAF inhibition.

View Article: PubMed Central - PubMed

Affiliation: Tumor Cell Biology Laboratory, Cancer Research UK London Research Institute, London WC2A 3LY, UK.

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ECMs and Rigid Substrates Mediate Drug Tolerance through Integrin β1-FAK Activation(A and B) 5555-mEGFP (in green) were seeded on polyacrylamide / bis-acrylamide gels with different stiffness (0.2, 3 and 12 kPa) coated with the indicated ECMs (ECM mixture contains FN, TNC, THBS1, and THBS2). Twenty-four hours after treatment with DMSO (0.1%) or PLX4720 (1 μM), cells are stained with propidium iodide (PI, in magenta). PI signals from the same experiments with 5555/4434-YFP-NLS were quantified in (B) (mean ± SD). Scale bars represent 100 μm.(C) 5555 cells cultured on the fibronectin-coated gels with the indicated stiffness were stained with a phospho-FAK antibody. The area of pFAK positive adhesion is quantified (mean ± SD). Scale bars represent 100 μm.(D) 5555 cells co-cultured with or without MAF1 on collagen gels were treated with DMSO (0.1%) or PLX4720 (1 μM) for 24 hr and stained with a phospho-FAK antibody. The area of pFAK positive adhesion in 5555 cell is quantified and shown as mean ± SD.(E) 5555-YFP-NLS (in green) transfected with the indicated siRNAs were co-cultured with MAF1 overnight. Then, cells were treated with DMSO (0.1%) or PLX4720 (1 μM) for 24 hr, followed by PI staining (in magenta). Scale bars represent 100 μm.(F) PI signals of (E) were quantified and shown as mean ± SD (G) PI signals from same experiments as in (E) using 4434 + MAF2 co-cultures were quantified and shown as mean ± SD.See also Figure S4.
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fig4: ECMs and Rigid Substrates Mediate Drug Tolerance through Integrin β1-FAK Activation(A and B) 5555-mEGFP (in green) were seeded on polyacrylamide / bis-acrylamide gels with different stiffness (0.2, 3 and 12 kPa) coated with the indicated ECMs (ECM mixture contains FN, TNC, THBS1, and THBS2). Twenty-four hours after treatment with DMSO (0.1%) or PLX4720 (1 μM), cells are stained with propidium iodide (PI, in magenta). PI signals from the same experiments with 5555/4434-YFP-NLS were quantified in (B) (mean ± SD). Scale bars represent 100 μm.(C) 5555 cells cultured on the fibronectin-coated gels with the indicated stiffness were stained with a phospho-FAK antibody. The area of pFAK positive adhesion is quantified (mean ± SD). Scale bars represent 100 μm.(D) 5555 cells co-cultured with or without MAF1 on collagen gels were treated with DMSO (0.1%) or PLX4720 (1 μM) for 24 hr and stained with a phospho-FAK antibody. The area of pFAK positive adhesion in 5555 cell is quantified and shown as mean ± SD.(E) 5555-YFP-NLS (in green) transfected with the indicated siRNAs were co-cultured with MAF1 overnight. Then, cells were treated with DMSO (0.1%) or PLX4720 (1 μM) for 24 hr, followed by PI staining (in magenta). Scale bars represent 100 μm.(F) PI signals of (E) were quantified and shown as mean ± SD (G) PI signals from same experiments as in (E) using 4434 + MAF2 co-cultures were quantified and shown as mean ± SD.See also Figure S4.

Mentions: The data above show that PLX4720 elicits changes in matrix production and remodeling by MAFs. To test if the ECM was sufficient to modulate the response of melanoma cells to PLX4720, we varied both matrix composition and matrix stiffness. Figure S4A shows that fibronectin (FN) consistently reduces the effect of PLX4720 on both 5555 and 4434 melanoma cells. Other matrix components, including THBS1/2 and TNC, have more variable effects between the two cell lines. In addition to matrix composition, matrix stiffness affects the behavior of cancer cells. Therefore, we combined variations in matrix composition and matrix stiffness. Polyacrylamide gels ranging from 0.2 kPa (similar to adipose tissue) to 12 kPa (similar to stiff tumors or muscle) were coated with either collagen I, FN, or a combination of FN, THBS1/2, and TNC. Melanoma cells on low stiffness collagen I showed high levels of cell death following BRAF inhibition (Figures 4A and 4B). However, this was greatly abrogated if cells were cultured on FN matrices with either 3 kPa or 12 kPa stiffness, with the most impressive cell survival on 12 kPa FN-, THBS1/2-, and TNC,containing matrices (Figures 4A and 4B). These data demonstrate that an appropriate matrix composition and stiffness can render BRAF-mutant melanoma cells insensitive to PLX4720.


Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin β1/FAK signaling.

Hirata E, Girotti MR, Viros A, Hooper S, Spencer-Dene B, Matsuda M, Larkin J, Marais R, Sahai E - Cancer Cell (2015)

ECMs and Rigid Substrates Mediate Drug Tolerance through Integrin β1-FAK Activation(A and B) 5555-mEGFP (in green) were seeded on polyacrylamide / bis-acrylamide gels with different stiffness (0.2, 3 and 12 kPa) coated with the indicated ECMs (ECM mixture contains FN, TNC, THBS1, and THBS2). Twenty-four hours after treatment with DMSO (0.1%) or PLX4720 (1 μM), cells are stained with propidium iodide (PI, in magenta). PI signals from the same experiments with 5555/4434-YFP-NLS were quantified in (B) (mean ± SD). Scale bars represent 100 μm.(C) 5555 cells cultured on the fibronectin-coated gels with the indicated stiffness were stained with a phospho-FAK antibody. The area of pFAK positive adhesion is quantified (mean ± SD). Scale bars represent 100 μm.(D) 5555 cells co-cultured with or without MAF1 on collagen gels were treated with DMSO (0.1%) or PLX4720 (1 μM) for 24 hr and stained with a phospho-FAK antibody. The area of pFAK positive adhesion in 5555 cell is quantified and shown as mean ± SD.(E) 5555-YFP-NLS (in green) transfected with the indicated siRNAs were co-cultured with MAF1 overnight. Then, cells were treated with DMSO (0.1%) or PLX4720 (1 μM) for 24 hr, followed by PI staining (in magenta). Scale bars represent 100 μm.(F) PI signals of (E) were quantified and shown as mean ± SD (G) PI signals from same experiments as in (E) using 4434 + MAF2 co-cultures were quantified and shown as mean ± SD.See also Figure S4.
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fig4: ECMs and Rigid Substrates Mediate Drug Tolerance through Integrin β1-FAK Activation(A and B) 5555-mEGFP (in green) were seeded on polyacrylamide / bis-acrylamide gels with different stiffness (0.2, 3 and 12 kPa) coated with the indicated ECMs (ECM mixture contains FN, TNC, THBS1, and THBS2). Twenty-four hours after treatment with DMSO (0.1%) or PLX4720 (1 μM), cells are stained with propidium iodide (PI, in magenta). PI signals from the same experiments with 5555/4434-YFP-NLS were quantified in (B) (mean ± SD). Scale bars represent 100 μm.(C) 5555 cells cultured on the fibronectin-coated gels with the indicated stiffness were stained with a phospho-FAK antibody. The area of pFAK positive adhesion is quantified (mean ± SD). Scale bars represent 100 μm.(D) 5555 cells co-cultured with or without MAF1 on collagen gels were treated with DMSO (0.1%) or PLX4720 (1 μM) for 24 hr and stained with a phospho-FAK antibody. The area of pFAK positive adhesion in 5555 cell is quantified and shown as mean ± SD.(E) 5555-YFP-NLS (in green) transfected with the indicated siRNAs were co-cultured with MAF1 overnight. Then, cells were treated with DMSO (0.1%) or PLX4720 (1 μM) for 24 hr, followed by PI staining (in magenta). Scale bars represent 100 μm.(F) PI signals of (E) were quantified and shown as mean ± SD (G) PI signals from same experiments as in (E) using 4434 + MAF2 co-cultures were quantified and shown as mean ± SD.See also Figure S4.
Mentions: The data above show that PLX4720 elicits changes in matrix production and remodeling by MAFs. To test if the ECM was sufficient to modulate the response of melanoma cells to PLX4720, we varied both matrix composition and matrix stiffness. Figure S4A shows that fibronectin (FN) consistently reduces the effect of PLX4720 on both 5555 and 4434 melanoma cells. Other matrix components, including THBS1/2 and TNC, have more variable effects between the two cell lines. In addition to matrix composition, matrix stiffness affects the behavior of cancer cells. Therefore, we combined variations in matrix composition and matrix stiffness. Polyacrylamide gels ranging from 0.2 kPa (similar to adipose tissue) to 12 kPa (similar to stiff tumors or muscle) were coated with either collagen I, FN, or a combination of FN, THBS1/2, and TNC. Melanoma cells on low stiffness collagen I showed high levels of cell death following BRAF inhibition (Figures 4A and 4B). However, this was greatly abrogated if cells were cultured on FN matrices with either 3 kPa or 12 kPa stiffness, with the most impressive cell survival on 12 kPa FN-, THBS1/2-, and TNC,containing matrices (Figures 4A and 4B). These data demonstrate that an appropriate matrix composition and stiffness can render BRAF-mutant melanoma cells insensitive to PLX4720.

Bottom Line: Intravital imaging of BRAF-mutant melanoma cells containing an ERK/MAPK biosensor reveals how the tumor microenvironment affects response to BRAF inhibition by PLX4720.Fibronectin-rich matrices with 3-12 kPa elastic modulus are sufficient to provide PLX4720 tolerance.We propose that paradoxically activated MAFs provide a "safe haven" for melanoma cells to tolerate BRAF inhibition.

View Article: PubMed Central - PubMed

Affiliation: Tumor Cell Biology Laboratory, Cancer Research UK London Research Institute, London WC2A 3LY, UK.

Show MeSH
Related in: MedlinePlus