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Role of mesenchymal stem cells in osteosarcoma and metabolic reprogramming of tumor cells.

Bonuccelli G, Avnet S, Grisendi G, Salerno M, Granchi D, Dominici M, Kusuzaki K, Baldini N - Oncotarget (2014)

Bottom Line: Our results showed that MSC are driven by oxidative stress induced by OS cells to undergo Warburg metabolism, with increased lactate production.Therefore, we analyzed the expression of lactate monocarboxylate transporters.We also showed that lactate produced by MSC promotes the migratory ability of OS cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Neuromotion Sciences, Alma Mater Studiorum-University of Bologna, Bologna, Italy.

ABSTRACT
The tumor microenvironment plays an important role in cancer progression. Here, we focused on the role of reactive mesenchymal stem cells (MSC) in osteosarcoma (OS), and used human adipose MSC and a panel of OS cell lines (Saos-2, HOS, and 143B) to investigate the mutual effect of normal-cancer cell metabolic programming. Our results showed that MSC are driven by oxidative stress induced by OS cells to undergo Warburg metabolism, with increased lactate production. Therefore, we analyzed the expression of lactate monocarboxylate transporters. By real time PCR and immunofluorescence, in MSC we detected the expression of MCT-4, the transporter for lactate efflux, whereas MCT-1, responsible for lactate uptake, was expressed in OS cells. In agreement, silencing of MCT-1 by siRNA significantly affected the ATP production in OS cancer cells. Thus, cancer cells directly increase their mitochondrial biogenesis using this energy-rich metabolite that is abundantly provided by MSC as an effect of the altered microenvironmental conditions induced by OS cells. We also showed that lactate produced by MSC promotes the migratory ability of OS cells. These data provide novel information to be exploited for cancer therapies targeting the mutual metabolic reprogramming of cancer cells and their stroma.

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Saos-2 cells drive aerobic glycolysis in adjacent MSC(A) Upregulation of PKM2 in MSC activated by OS cells. Cell lysates were prepared from MSC activated by conditioned medium from serum-starved Saos-2 cells or untreated, non-activated MSC. Lysates were analyzed by Western blot with an anti-PKM2 specific antibody. Note the upregulation of PKM2 in tumor-activated MSC, as compared to untreated MSC. Normalization was done by actin immunoblot. (B) Gene expression analysis of GLUT1 (left plot) and MCT-4 (right plot) by RT-PCR. Note that MSC cultured with CM from Saos-2 cells, show significantly higher levels of the GLUT1 and MCT-4 genes, versus non-activated MSC. Glut1, p=0.0092; MCT-4, p=0.0071. (C) Upregulation of MCT-4 expression was validated by immunofluorescence analysis. Homotypic cultures of MSC (top row) and Saos-2 GFP cells (middle row) and Saos-2-MSC heterotypic cultures (lower row) were immunostained with MCT-4 antibodies. DAPI was used to stain nuclei (blue). Note that the MCT-4 expression (red) is clearly increased in MSC in co-culture condition, as compared to MSC cultured alone. Importantly, images were acquired using identical exposure settings. Original magnification, 20x. Scale bar 40 μm. (D) Tumor-activated MSC show increased lactate secretion. MSC were incubated with CM obtained from OS cells (Saos-2 or HOS) for 48 hours. Then, cells were carefully washed and incubated in fresh serum-free medium for an additional 24 hours. Lactate assay was performed on this culture medium. Note that lactate production is significantly increased in MSC, after activation with conditioned media from Saos-2 cells (left plot) and HOS cells (right plot). Values were normalized by cell numbers *p<0.05.
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Figure 2: Saos-2 cells drive aerobic glycolysis in adjacent MSC(A) Upregulation of PKM2 in MSC activated by OS cells. Cell lysates were prepared from MSC activated by conditioned medium from serum-starved Saos-2 cells or untreated, non-activated MSC. Lysates were analyzed by Western blot with an anti-PKM2 specific antibody. Note the upregulation of PKM2 in tumor-activated MSC, as compared to untreated MSC. Normalization was done by actin immunoblot. (B) Gene expression analysis of GLUT1 (left plot) and MCT-4 (right plot) by RT-PCR. Note that MSC cultured with CM from Saos-2 cells, show significantly higher levels of the GLUT1 and MCT-4 genes, versus non-activated MSC. Glut1, p=0.0092; MCT-4, p=0.0071. (C) Upregulation of MCT-4 expression was validated by immunofluorescence analysis. Homotypic cultures of MSC (top row) and Saos-2 GFP cells (middle row) and Saos-2-MSC heterotypic cultures (lower row) were immunostained with MCT-4 antibodies. DAPI was used to stain nuclei (blue). Note that the MCT-4 expression (red) is clearly increased in MSC in co-culture condition, as compared to MSC cultured alone. Importantly, images were acquired using identical exposure settings. Original magnification, 20x. Scale bar 40 μm. (D) Tumor-activated MSC show increased lactate secretion. MSC were incubated with CM obtained from OS cells (Saos-2 or HOS) for 48 hours. Then, cells were carefully washed and incubated in fresh serum-free medium for an additional 24 hours. Lactate assay was performed on this culture medium. Note that lactate production is significantly increased in MSC, after activation with conditioned media from Saos-2 cells (left plot) and HOS cells (right plot). Values were normalized by cell numbers *p<0.05.

Mentions: Next, we set out to investigate the glycolytic profile of MSC. To this end, we studied the expression of pyruvate kinase isoform 2 (PKM2), a key regulator of glycolysis and promoter of tumor growth [19]. For this purpose, MSC were incubated with conditioned medium (CM) derived from Saos-2 cells for 24 hours. Western blot analysis demonstrates that MSC activated by Saos-2-CM have a higher PKM2 expression, as compared to untreated MSC (Figure 2A), suggesting that the conditioned medium from Saos-2 cells induces a glycolytic switch in MSC. A shift towards a glycolytic phenotype is also supported by the real time PCR analysis for the glucose transporter GLUT1 mRNA in CM-activated MSC versus not activated MSC. Figure 2B (left plot) shows increased GLUT1 expression in MSC after activation with Saos-2-CM, suggesting an enhanced glucose uptake and increased glycolysis.


Role of mesenchymal stem cells in osteosarcoma and metabolic reprogramming of tumor cells.

Bonuccelli G, Avnet S, Grisendi G, Salerno M, Granchi D, Dominici M, Kusuzaki K, Baldini N - Oncotarget (2014)

Saos-2 cells drive aerobic glycolysis in adjacent MSC(A) Upregulation of PKM2 in MSC activated by OS cells. Cell lysates were prepared from MSC activated by conditioned medium from serum-starved Saos-2 cells or untreated, non-activated MSC. Lysates were analyzed by Western blot with an anti-PKM2 specific antibody. Note the upregulation of PKM2 in tumor-activated MSC, as compared to untreated MSC. Normalization was done by actin immunoblot. (B) Gene expression analysis of GLUT1 (left plot) and MCT-4 (right plot) by RT-PCR. Note that MSC cultured with CM from Saos-2 cells, show significantly higher levels of the GLUT1 and MCT-4 genes, versus non-activated MSC. Glut1, p=0.0092; MCT-4, p=0.0071. (C) Upregulation of MCT-4 expression was validated by immunofluorescence analysis. Homotypic cultures of MSC (top row) and Saos-2 GFP cells (middle row) and Saos-2-MSC heterotypic cultures (lower row) were immunostained with MCT-4 antibodies. DAPI was used to stain nuclei (blue). Note that the MCT-4 expression (red) is clearly increased in MSC in co-culture condition, as compared to MSC cultured alone. Importantly, images were acquired using identical exposure settings. Original magnification, 20x. Scale bar 40 μm. (D) Tumor-activated MSC show increased lactate secretion. MSC were incubated with CM obtained from OS cells (Saos-2 or HOS) for 48 hours. Then, cells were carefully washed and incubated in fresh serum-free medium for an additional 24 hours. Lactate assay was performed on this culture medium. Note that lactate production is significantly increased in MSC, after activation with conditioned media from Saos-2 cells (left plot) and HOS cells (right plot). Values were normalized by cell numbers *p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Saos-2 cells drive aerobic glycolysis in adjacent MSC(A) Upregulation of PKM2 in MSC activated by OS cells. Cell lysates were prepared from MSC activated by conditioned medium from serum-starved Saos-2 cells or untreated, non-activated MSC. Lysates were analyzed by Western blot with an anti-PKM2 specific antibody. Note the upregulation of PKM2 in tumor-activated MSC, as compared to untreated MSC. Normalization was done by actin immunoblot. (B) Gene expression analysis of GLUT1 (left plot) and MCT-4 (right plot) by RT-PCR. Note that MSC cultured with CM from Saos-2 cells, show significantly higher levels of the GLUT1 and MCT-4 genes, versus non-activated MSC. Glut1, p=0.0092; MCT-4, p=0.0071. (C) Upregulation of MCT-4 expression was validated by immunofluorescence analysis. Homotypic cultures of MSC (top row) and Saos-2 GFP cells (middle row) and Saos-2-MSC heterotypic cultures (lower row) were immunostained with MCT-4 antibodies. DAPI was used to stain nuclei (blue). Note that the MCT-4 expression (red) is clearly increased in MSC in co-culture condition, as compared to MSC cultured alone. Importantly, images were acquired using identical exposure settings. Original magnification, 20x. Scale bar 40 μm. (D) Tumor-activated MSC show increased lactate secretion. MSC were incubated with CM obtained from OS cells (Saos-2 or HOS) for 48 hours. Then, cells were carefully washed and incubated in fresh serum-free medium for an additional 24 hours. Lactate assay was performed on this culture medium. Note that lactate production is significantly increased in MSC, after activation with conditioned media from Saos-2 cells (left plot) and HOS cells (right plot). Values were normalized by cell numbers *p<0.05.
Mentions: Next, we set out to investigate the glycolytic profile of MSC. To this end, we studied the expression of pyruvate kinase isoform 2 (PKM2), a key regulator of glycolysis and promoter of tumor growth [19]. For this purpose, MSC were incubated with conditioned medium (CM) derived from Saos-2 cells for 24 hours. Western blot analysis demonstrates that MSC activated by Saos-2-CM have a higher PKM2 expression, as compared to untreated MSC (Figure 2A), suggesting that the conditioned medium from Saos-2 cells induces a glycolytic switch in MSC. A shift towards a glycolytic phenotype is also supported by the real time PCR analysis for the glucose transporter GLUT1 mRNA in CM-activated MSC versus not activated MSC. Figure 2B (left plot) shows increased GLUT1 expression in MSC after activation with Saos-2-CM, suggesting an enhanced glucose uptake and increased glycolysis.

Bottom Line: Our results showed that MSC are driven by oxidative stress induced by OS cells to undergo Warburg metabolism, with increased lactate production.Therefore, we analyzed the expression of lactate monocarboxylate transporters.We also showed that lactate produced by MSC promotes the migratory ability of OS cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Neuromotion Sciences, Alma Mater Studiorum-University of Bologna, Bologna, Italy.

ABSTRACT
The tumor microenvironment plays an important role in cancer progression. Here, we focused on the role of reactive mesenchymal stem cells (MSC) in osteosarcoma (OS), and used human adipose MSC and a panel of OS cell lines (Saos-2, HOS, and 143B) to investigate the mutual effect of normal-cancer cell metabolic programming. Our results showed that MSC are driven by oxidative stress induced by OS cells to undergo Warburg metabolism, with increased lactate production. Therefore, we analyzed the expression of lactate monocarboxylate transporters. By real time PCR and immunofluorescence, in MSC we detected the expression of MCT-4, the transporter for lactate efflux, whereas MCT-1, responsible for lactate uptake, was expressed in OS cells. In agreement, silencing of MCT-1 by siRNA significantly affected the ATP production in OS cancer cells. Thus, cancer cells directly increase their mitochondrial biogenesis using this energy-rich metabolite that is abundantly provided by MSC as an effect of the altered microenvironmental conditions induced by OS cells. We also showed that lactate produced by MSC promotes the migratory ability of OS cells. These data provide novel information to be exploited for cancer therapies targeting the mutual metabolic reprogramming of cancer cells and their stroma.

Show MeSH
Related in: MedlinePlus