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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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Related in: MedlinePlus

Stromal Cells Provide Invasive and Pro-survival Signals, and the System Adapts to the Drug Within 12 Hours(A and B) Various types (indicated above in each panel) of 5555 melanoma spheroids/tumor explants were embedded into collagen gels and treated with 0.1% DMSO (upper panels) or 1 μM PLX4720 (lower panels) for 24 hr. 5555 cells are depicted in green, stromal cells/MAFs in magenta, and representative DAPI-stained images are also shown (A), and the ratio of DAPI-positive debris and melanoma nucleus was calculated as a cell death indicator (B). Spheroid, 5555 pure spheroids made in vitro; explant, tumor explants of 5555 grown subcutaneously in C57BL/6_ROSA26-mTmG mice; lung mets, tumor explants of experimentally-induced lung metastasis of 5555 in C57BL/6_ROSA26-mTmG mice; +MAF1/2, 5555 and MAF co-culture spheroid made in vitro; +MAF1/2-CM, 5555 spheroids made in vitro were treated with conditioned media from MAFs. Data in (B) are represented as mean ± SD. Scale bars represent 100 μm.(C–E) 5555/4434-EKAREV-NLS and MAF1/2-mCherry co-cultured spheroids were embedded in collagen gels and treated with PLX4720 (1 μM) at time = 0. Upper images in (C) indicate 5555 nuclei (gray) and MAF2 (magenta), and lower images indicate ERK activity (FRET/CFP) in 5555 cells. Kymographs of ERK activity in seven representative cells are shown in (D), and ERK activities at time −30 min, +30 min, +4 hr, and +12 hr are quantified and shown in (E) as scatterplots with mean ± SD. Scale bar represents 100 μm.(F and G) 5555/4434-EKAREV-NLS and MAF1/2 co-cultured spheroids in collagen gels were treated with PLX4720 (1 μM) for 12 hr, followed by re-treatment with PLX4720 (1 μM) or PD184352 (1 μM). Representative images of 5555 co-cultured with MAF1 are shown in (F) and ERK activities (FRET/CFP) in 5555 and 4434 before and after re-treatment were quantified and shown as scatterplots with mean ± SD (G).Scale bars represent 100 μm. See also Figure S2 and Movie S1.
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fig2: Stromal Cells Provide Invasive and Pro-survival Signals, and the System Adapts to the Drug Within 12 Hours(A and B) Various types (indicated above in each panel) of 5555 melanoma spheroids/tumor explants were embedded into collagen gels and treated with 0.1% DMSO (upper panels) or 1 μM PLX4720 (lower panels) for 24 hr. 5555 cells are depicted in green, stromal cells/MAFs in magenta, and representative DAPI-stained images are also shown (A), and the ratio of DAPI-positive debris and melanoma nucleus was calculated as a cell death indicator (B). Spheroid, 5555 pure spheroids made in vitro; explant, tumor explants of 5555 grown subcutaneously in C57BL/6_ROSA26-mTmG mice; lung mets, tumor explants of experimentally-induced lung metastasis of 5555 in C57BL/6_ROSA26-mTmG mice; +MAF1/2, 5555 and MAF co-culture spheroid made in vitro; +MAF1/2-CM, 5555 spheroids made in vitro were treated with conditioned media from MAFs. Data in (B) are represented as mean ± SD. Scale bars represent 100 μm.(C–E) 5555/4434-EKAREV-NLS and MAF1/2-mCherry co-cultured spheroids were embedded in collagen gels and treated with PLX4720 (1 μM) at time = 0. Upper images in (C) indicate 5555 nuclei (gray) and MAF2 (magenta), and lower images indicate ERK activity (FRET/CFP) in 5555 cells. Kymographs of ERK activity in seven representative cells are shown in (D), and ERK activities at time −30 min, +30 min, +4 hr, and +12 hr are quantified and shown in (E) as scatterplots with mean ± SD. Scale bar represents 100 μm.(F and G) 5555/4434-EKAREV-NLS and MAF1/2 co-cultured spheroids in collagen gels were treated with PLX4720 (1 μM) for 12 hr, followed by re-treatment with PLX4720 (1 μM) or PD184352 (1 μM). Representative images of 5555 co-cultured with MAF1 are shown in (F) and ERK activities (FRET/CFP) in 5555 and 4434 before and after re-treatment were quantified and shown as scatterplots with mean ± SD (G).Scale bars represent 100 μm. See also Figure S2 and Movie S1.

Mentions: We sought to establish a culture system that re-capitulated the PLX4720 tolerance that we observed in vivo. The behavior of pure spheroids of melanoma cells was compared with that of equivalent sized tumor pieces when embedded in a collagen matrix. Figures 2A and 2B show that pure melanoma spheroids were highly sensitive to PLX4720, with the appearance of many nuclear fragments indicating cell death. In contrast, melanoma explants from either subcutaneous tumors or experimentally established lung metastases were unresponsive to PLX4720 (Figures 2A and 2B). Even in the presence of drug, melanoma cells retained healthy nuclear architecture and invasive capability. Thus, the spheroid model recapitulates the stroma-dependent PLX4720 tolerance observed in vivo. It also formally excludes any problems relating to drug access that might confound interpretation of the in vivo data.


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)

Stromal Cells Provide Invasive and Pro-survival Signals, and the System Adapts to the Drug Within 12 Hours(A and B) Various types (indicated above in each panel) of 5555 melanoma spheroids/tumor explants were embedded into collagen gels and treated with 0.1% DMSO (upper panels) or 1 μM PLX4720 (lower panels) for 24 hr. 5555 cells are depicted in green, stromal cells/MAFs in magenta, and representative DAPI-stained images are also shown (A), and the ratio of DAPI-positive debris and melanoma nucleus was calculated as a cell death indicator (B). Spheroid, 5555 pure spheroids made in vitro; explant, tumor explants of 5555 grown subcutaneously in C57BL/6_ROSA26-mTmG mice; lung mets, tumor explants of experimentally-induced lung metastasis of 5555 in C57BL/6_ROSA26-mTmG mice; +MAF1/2, 5555 and MAF co-culture spheroid made in vitro; +MAF1/2-CM, 5555 spheroids made in vitro were treated with conditioned media from MAFs. Data in (B) are represented as mean ± SD. Scale bars represent 100 μm.(C–E) 5555/4434-EKAREV-NLS and MAF1/2-mCherry co-cultured spheroids were embedded in collagen gels and treated with PLX4720 (1 μM) at time = 0. Upper images in (C) indicate 5555 nuclei (gray) and MAF2 (magenta), and lower images indicate ERK activity (FRET/CFP) in 5555 cells. Kymographs of ERK activity in seven representative cells are shown in (D), and ERK activities at time −30 min, +30 min, +4 hr, and +12 hr are quantified and shown in (E) as scatterplots with mean ± SD. Scale bar represents 100 μm.(F and G) 5555/4434-EKAREV-NLS and MAF1/2 co-cultured spheroids in collagen gels were treated with PLX4720 (1 μM) for 12 hr, followed by re-treatment with PLX4720 (1 μM) or PD184352 (1 μM). Representative images of 5555 co-cultured with MAF1 are shown in (F) and ERK activities (FRET/CFP) in 5555 and 4434 before and after re-treatment were quantified and shown as scatterplots with mean ± SD (G).Scale bars represent 100 μm. See also Figure S2 and Movie S1.
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fig2: Stromal Cells Provide Invasive and Pro-survival Signals, and the System Adapts to the Drug Within 12 Hours(A and B) Various types (indicated above in each panel) of 5555 melanoma spheroids/tumor explants were embedded into collagen gels and treated with 0.1% DMSO (upper panels) or 1 μM PLX4720 (lower panels) for 24 hr. 5555 cells are depicted in green, stromal cells/MAFs in magenta, and representative DAPI-stained images are also shown (A), and the ratio of DAPI-positive debris and melanoma nucleus was calculated as a cell death indicator (B). Spheroid, 5555 pure spheroids made in vitro; explant, tumor explants of 5555 grown subcutaneously in C57BL/6_ROSA26-mTmG mice; lung mets, tumor explants of experimentally-induced lung metastasis of 5555 in C57BL/6_ROSA26-mTmG mice; +MAF1/2, 5555 and MAF co-culture spheroid made in vitro; +MAF1/2-CM, 5555 spheroids made in vitro were treated with conditioned media from MAFs. Data in (B) are represented as mean ± SD. Scale bars represent 100 μm.(C–E) 5555/4434-EKAREV-NLS and MAF1/2-mCherry co-cultured spheroids were embedded in collagen gels and treated with PLX4720 (1 μM) at time = 0. Upper images in (C) indicate 5555 nuclei (gray) and MAF2 (magenta), and lower images indicate ERK activity (FRET/CFP) in 5555 cells. Kymographs of ERK activity in seven representative cells are shown in (D), and ERK activities at time −30 min, +30 min, +4 hr, and +12 hr are quantified and shown in (E) as scatterplots with mean ± SD. Scale bar represents 100 μm.(F and G) 5555/4434-EKAREV-NLS and MAF1/2 co-cultured spheroids in collagen gels were treated with PLX4720 (1 μM) for 12 hr, followed by re-treatment with PLX4720 (1 μM) or PD184352 (1 μM). Representative images of 5555 co-cultured with MAF1 are shown in (F) and ERK activities (FRET/CFP) in 5555 and 4434 before and after re-treatment were quantified and shown as scatterplots with mean ± SD (G).Scale bars represent 100 μm. See also Figure S2 and Movie S1.
Mentions: We sought to establish a culture system that re-capitulated the PLX4720 tolerance that we observed in vivo. The behavior of pure spheroids of melanoma cells was compared with that of equivalent sized tumor pieces when embedded in a collagen matrix. Figures 2A and 2B show that pure melanoma spheroids were highly sensitive to PLX4720, with the appearance of many nuclear fragments indicating cell death. In contrast, melanoma explants from either subcutaneous tumors or experimentally established lung metastases were unresponsive to PLX4720 (Figures 2A and 2B). Even in the presence of drug, melanoma cells retained healthy nuclear architecture and invasive capability. Thus, the spheroid model recapitulates the stroma-dependent PLX4720 tolerance observed in vivo. It also formally excludes any problems relating to drug access that might confound interpretation of the in vivo data.

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