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Skeletal muscle phenotypically converts and selectively inhibits metastatic cells in mice.

Parlakian A, Gomaa I, Solly S, Arandel L, Mahale A, Born G, Marazzi G, Sassoon D - PLoS ONE (2010)

Bottom Line: No effect is seen with non-tumorigenic cells.Tumor suppression assays reveal that the muscle-mediated tumor suppressor effects do not generate resistant clones but function through the down-regulation of the transcription factor MiTF, a master regulator of melanocyte development and a melanoma oncogene.Our findings point to skeletal muscle as a source of therapeutic agents in the treatment of metastatic cancers.

View Article: PubMed Central - PubMed

Affiliation: Myology Group, UMR S 787 Inserm, Université Paris VI/Pierre et Marie Curie, Paris, France.

ABSTRACT
Skeletal muscle is rarely a site of malignant metastasis; the molecular and cellular basis for this rarity is not understood. We report that myogenic cells exert pronounced effects upon co-culture with metastatic melanoma (B16-F10) or carcinoma (LLC1) cells including conversion to the myogenic lineage in vitro and in vivo, as well as inhibition of melanin production in melanoma cells coupled with cytotoxic and cytostatic effects. No effect is seen with non-tumorigenic cells. Tumor suppression assays reveal that the muscle-mediated tumor suppressor effects do not generate resistant clones but function through the down-regulation of the transcription factor MiTF, a master regulator of melanocyte development and a melanoma oncogene. Our findings point to skeletal muscle as a source of therapeutic agents in the treatment of metastatic cancers.

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Apoptotic effect of the conditioned media from C2C12 muscle cells on B16 melanoma cells.A,B, upper panels) Phase contrast photomicrographs of 10T1/2 fibroblasts (A) and B16-F10 melanoma cells (B) cultured in serum free conditioned media from 10T1/2 fibroblasts (CM10T1/2) or in serum free conditioned media from muscle cells (CMC2C12) for 3 days. After 3 days in CMC2C12, B16 melanoma cells are less numerous and appear rounded and refractive (B). Scale bar  = 50 µm. A,B, lower panels) Propidium iodide flow cytometry cell cycle analysis of 10T1/2 fibroblasts (A) and B16 melanoma cells (B) in the different culture media. The bars indicate the fraction of apoptotic cells for each experimental condition. The cell cycle profile is notably changed in melanoma cells incubated for 3 days in muscle cells conditioned media. C) Percentage of apoptotic versus cycling cells of 10T1/2 fibroblasts (blue bars) and B16-F10 melanoma cells (green bars) cultured in either serum free conditioned media from 10T1/2 fibroblast (CM10T1/2) or C2C12 muscle cells (CMC2C12) after 1 and 3 days post incubation (dpi). Only B16F10 melanoma cells exhibit a significantly high level of apoptotic cells (40.5%±2.5) when cultured in CMC2C12 for 3 days (p≤0.05). Values represent the mean % ± SEM of 3 independent experiments.
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pone-0009299-g004: Apoptotic effect of the conditioned media from C2C12 muscle cells on B16 melanoma cells.A,B, upper panels) Phase contrast photomicrographs of 10T1/2 fibroblasts (A) and B16-F10 melanoma cells (B) cultured in serum free conditioned media from 10T1/2 fibroblasts (CM10T1/2) or in serum free conditioned media from muscle cells (CMC2C12) for 3 days. After 3 days in CMC2C12, B16 melanoma cells are less numerous and appear rounded and refractive (B). Scale bar  = 50 µm. A,B, lower panels) Propidium iodide flow cytometry cell cycle analysis of 10T1/2 fibroblasts (A) and B16 melanoma cells (B) in the different culture media. The bars indicate the fraction of apoptotic cells for each experimental condition. The cell cycle profile is notably changed in melanoma cells incubated for 3 days in muscle cells conditioned media. C) Percentage of apoptotic versus cycling cells of 10T1/2 fibroblasts (blue bars) and B16-F10 melanoma cells (green bars) cultured in either serum free conditioned media from 10T1/2 fibroblast (CM10T1/2) or C2C12 muscle cells (CMC2C12) after 1 and 3 days post incubation (dpi). Only B16F10 melanoma cells exhibit a significantly high level of apoptotic cells (40.5%±2.5) when cultured in CMC2C12 for 3 days (p≤0.05). Values represent the mean % ± SEM of 3 independent experiments.

Mentions: Co-culture experiments with B16-GFP or LLC1-GFP and differentiating C2C12 cells gave rise to cultures which appeared to contain less tumor cells (Figure 1A and Figure S1). It is well accepted that acquired resistance to apoptotic and/or growth arrest signals plays a major role in tumor progression [50], [51]. To determine if diffusible factors secreted by muscle cells induce cell cycle arrest and/or cell death in B16 cells, we collected serum-free conditioned media from C2C12 muscle cultures (CMC2C12) and from 10T1/2 fibroblast cultures (CM10T1/2). We tested conditioned media on B16 cells as well as C2C12 myoblasts and 10T1/2 fibroblasts and monitored cell morphology, number, and cell cycle status using propidium iodide uptake followed by FACs analysis. 10T1/2 fibroblasts grown in CM10T1/2 or CMC2C12 display robust growth and do not display overt changes in morphology (Figure 4A, upper panels). Furthermore, we did not observe significant differences in cell cycle profiles of 10T1/2 fibroblasts exposed to CM10T1/2 or CMC2C12 (Figure 4A, lower panels). After one day in fibroblasts conditioned media (CM10T1/2) 0,6% of the 10T1/2 fibroblasts were apoptotic versus 2,5% of apoptotic cells in cultures incubated in conditioned media from myogenic cells (CMC2C12) (Figure 4C). The percentage of apoptotic fibroblast cells did not vary 3 days after incubation in both conditioned media (Figure 4C). When B16 melanoma cells were incubated for 1 day in CM10T1/2 or in CMC2C12, we do not observe a significant change in the cell morphology (data not shown) nor in cell cycle profile (Figure 4C). There were no changes in the cell morphology of B16 cells grown for 3 days in fibroblast conditioned media (Figure 4B, upper panels) and the percentage of apoptotic B16 melanoma cells did not vary significantly between 1 day or 3 days of exposure to CM10T1/2 (Figure 4B and C). In contrast, B16 melanoma cells incubated in (CMC2C12) for 3 days appear refractile and rounded (Figure 4B, upper panels). In addition, the cell cycle profiles show a marked increase in the percentage of apoptotic cells (40,5% versus 59,5% of cycling cells) (Figure 4B, C). The apoptotic effect of muscle conditioned media was not restricted to the B16 melanoma cells as we obtained similar results with Lewis-Lung carcinoma cells (Figure S2) indicating a specific effect by muscle conditioned media upon at least 2 metastatic cell types. As expected, CM10T1/2 did not have any apoptotic effect on the Lewis-Lung carcinoma cells (Figure S2).


Skeletal muscle phenotypically converts and selectively inhibits metastatic cells in mice.

Parlakian A, Gomaa I, Solly S, Arandel L, Mahale A, Born G, Marazzi G, Sassoon D - PLoS ONE (2010)

Apoptotic effect of the conditioned media from C2C12 muscle cells on B16 melanoma cells.A,B, upper panels) Phase contrast photomicrographs of 10T1/2 fibroblasts (A) and B16-F10 melanoma cells (B) cultured in serum free conditioned media from 10T1/2 fibroblasts (CM10T1/2) or in serum free conditioned media from muscle cells (CMC2C12) for 3 days. After 3 days in CMC2C12, B16 melanoma cells are less numerous and appear rounded and refractive (B). Scale bar  = 50 µm. A,B, lower panels) Propidium iodide flow cytometry cell cycle analysis of 10T1/2 fibroblasts (A) and B16 melanoma cells (B) in the different culture media. The bars indicate the fraction of apoptotic cells for each experimental condition. The cell cycle profile is notably changed in melanoma cells incubated for 3 days in muscle cells conditioned media. C) Percentage of apoptotic versus cycling cells of 10T1/2 fibroblasts (blue bars) and B16-F10 melanoma cells (green bars) cultured in either serum free conditioned media from 10T1/2 fibroblast (CM10T1/2) or C2C12 muscle cells (CMC2C12) after 1 and 3 days post incubation (dpi). Only B16F10 melanoma cells exhibit a significantly high level of apoptotic cells (40.5%±2.5) when cultured in CMC2C12 for 3 days (p≤0.05). Values represent the mean % ± SEM of 3 independent experiments.
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pone-0009299-g004: Apoptotic effect of the conditioned media from C2C12 muscle cells on B16 melanoma cells.A,B, upper panels) Phase contrast photomicrographs of 10T1/2 fibroblasts (A) and B16-F10 melanoma cells (B) cultured in serum free conditioned media from 10T1/2 fibroblasts (CM10T1/2) or in serum free conditioned media from muscle cells (CMC2C12) for 3 days. After 3 days in CMC2C12, B16 melanoma cells are less numerous and appear rounded and refractive (B). Scale bar  = 50 µm. A,B, lower panels) Propidium iodide flow cytometry cell cycle analysis of 10T1/2 fibroblasts (A) and B16 melanoma cells (B) in the different culture media. The bars indicate the fraction of apoptotic cells for each experimental condition. The cell cycle profile is notably changed in melanoma cells incubated for 3 days in muscle cells conditioned media. C) Percentage of apoptotic versus cycling cells of 10T1/2 fibroblasts (blue bars) and B16-F10 melanoma cells (green bars) cultured in either serum free conditioned media from 10T1/2 fibroblast (CM10T1/2) or C2C12 muscle cells (CMC2C12) after 1 and 3 days post incubation (dpi). Only B16F10 melanoma cells exhibit a significantly high level of apoptotic cells (40.5%±2.5) when cultured in CMC2C12 for 3 days (p≤0.05). Values represent the mean % ± SEM of 3 independent experiments.
Mentions: Co-culture experiments with B16-GFP or LLC1-GFP and differentiating C2C12 cells gave rise to cultures which appeared to contain less tumor cells (Figure 1A and Figure S1). It is well accepted that acquired resistance to apoptotic and/or growth arrest signals plays a major role in tumor progression [50], [51]. To determine if diffusible factors secreted by muscle cells induce cell cycle arrest and/or cell death in B16 cells, we collected serum-free conditioned media from C2C12 muscle cultures (CMC2C12) and from 10T1/2 fibroblast cultures (CM10T1/2). We tested conditioned media on B16 cells as well as C2C12 myoblasts and 10T1/2 fibroblasts and monitored cell morphology, number, and cell cycle status using propidium iodide uptake followed by FACs analysis. 10T1/2 fibroblasts grown in CM10T1/2 or CMC2C12 display robust growth and do not display overt changes in morphology (Figure 4A, upper panels). Furthermore, we did not observe significant differences in cell cycle profiles of 10T1/2 fibroblasts exposed to CM10T1/2 or CMC2C12 (Figure 4A, lower panels). After one day in fibroblasts conditioned media (CM10T1/2) 0,6% of the 10T1/2 fibroblasts were apoptotic versus 2,5% of apoptotic cells in cultures incubated in conditioned media from myogenic cells (CMC2C12) (Figure 4C). The percentage of apoptotic fibroblast cells did not vary 3 days after incubation in both conditioned media (Figure 4C). When B16 melanoma cells were incubated for 1 day in CM10T1/2 or in CMC2C12, we do not observe a significant change in the cell morphology (data not shown) nor in cell cycle profile (Figure 4C). There were no changes in the cell morphology of B16 cells grown for 3 days in fibroblast conditioned media (Figure 4B, upper panels) and the percentage of apoptotic B16 melanoma cells did not vary significantly between 1 day or 3 days of exposure to CM10T1/2 (Figure 4B and C). In contrast, B16 melanoma cells incubated in (CMC2C12) for 3 days appear refractile and rounded (Figure 4B, upper panels). In addition, the cell cycle profiles show a marked increase in the percentage of apoptotic cells (40,5% versus 59,5% of cycling cells) (Figure 4B, C). The apoptotic effect of muscle conditioned media was not restricted to the B16 melanoma cells as we obtained similar results with Lewis-Lung carcinoma cells (Figure S2) indicating a specific effect by muscle conditioned media upon at least 2 metastatic cell types. As expected, CM10T1/2 did not have any apoptotic effect on the Lewis-Lung carcinoma cells (Figure S2).

Bottom Line: No effect is seen with non-tumorigenic cells.Tumor suppression assays reveal that the muscle-mediated tumor suppressor effects do not generate resistant clones but function through the down-regulation of the transcription factor MiTF, a master regulator of melanocyte development and a melanoma oncogene.Our findings point to skeletal muscle as a source of therapeutic agents in the treatment of metastatic cancers.

View Article: PubMed Central - PubMed

Affiliation: Myology Group, UMR S 787 Inserm, Université Paris VI/Pierre et Marie Curie, Paris, France.

ABSTRACT
Skeletal muscle is rarely a site of malignant metastasis; the molecular and cellular basis for this rarity is not understood. We report that myogenic cells exert pronounced effects upon co-culture with metastatic melanoma (B16-F10) or carcinoma (LLC1) cells including conversion to the myogenic lineage in vitro and in vivo, as well as inhibition of melanin production in melanoma cells coupled with cytotoxic and cytostatic effects. No effect is seen with non-tumorigenic cells. Tumor suppression assays reveal that the muscle-mediated tumor suppressor effects do not generate resistant clones but function through the down-regulation of the transcription factor MiTF, a master regulator of melanocyte development and a melanoma oncogene. Our findings point to skeletal muscle as a source of therapeutic agents in the treatment of metastatic cancers.

Show MeSH
Related in: MedlinePlus