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Stimulation of hepatocarcinogenesis by neutrophils upon induction of oncogenic kras expression in transgenic zebrafish.

Yan C, Huo X, Wang S, Feng Y, Gong Z - J. Hepatol. (2015)

Bottom Line: Both oncogenic hepatocytes and tumor-associated neutrophils (TANs) were isolated via fluorescence-activated cell sorting.Molecular analyses indicated a pro-inflammatory microenvironment, as marked by increased tgfβ1a expression in kras(V12)-expressing hepatocytes and a loss of anti-tumor activities in TANs.Depletion of Tgf-β significantly reduced the number of TANs and the size of oncogenic liver.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore; National University of Singapore Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore.

No MeSH data available.


Related in: MedlinePlus

Effect of infiltrated neutrophils on liver size. (A–C) Neutrophil counts (left) and density (middle) in the liver area and liver size (right) in response to treatments of LSP (A), FPR A14 (B) or PR39 (C). Both kras−/lyz+ and kras+/lyz+ groups were similarly exposed to doxycycline from 3 dpf and neutrophils mediators were added from 4 dpf. Neutrophils and liver sizes were determined at 8 dpf (n >15 from each group). Statistical significance: *p <0.05.
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f0010: Effect of infiltrated neutrophils on liver size. (A–C) Neutrophil counts (left) and density (middle) in the liver area and liver size (right) in response to treatments of LSP (A), FPR A14 (B) or PR39 (C). Both kras−/lyz+ and kras+/lyz+ groups were similarly exposed to doxycycline from 3 dpf and neutrophils mediators were added from 4 dpf. Neutrophils and liver sizes were determined at 8 dpf (n >15 from each group). Statistical significance: *p <0.05.

Mentions: To further demonstrate the role of inflammatory cells in initiation and progression of hapatocarcinogenesis, we first tested a general inflammatory stimulator, LPS, which has been demonstrated to stimulate the immune system in zebrafish larvae [26]. 5 ng/ml of LPS was used to treat zebrafish larvae from 4 dpf to 8 dpf. kras+ larvae exposed to both LPS and doxycycline showed significant increases of both neutrophil count and density in the liver as compared to kras+ larvae exposed to doxycycline alone and all kras− groups. Interestingly, there was also a further enlargement of liver size with the increased neutrophils (Fig. 2A). To investigate if the accelerated liver enlargement was indeed associated with increased neutrophil infiltration, FPR-A14, which is a formyl peptide receptor agonist and has been reported to potently activate neutrophils specifically in vitro[27], was used to challenge the kras+ larvae from 4 dpf to 8 dpf. Liver neutrophil count and density in FPR-A14 and doxycycline double-treated kras+ larvae were also significantly higher than those of their kras+ sibling treated with only doxycycline and of all kras− groups (Fig. 2B), similar to that observed following LPS treatment. A further liver enlargement was also observed from these double-treated kras+ larvae. To further demonstrate the effect of neutrophils, kras+ transgenic larvae were also challenged with a neutrophil inhibitor, PR-39, a proline-rich anti-bacteria peptide that inhibits NADPH oxidase activity in neutrophils [28]. As shown in Fig. 2C, liver neutrophil count and density as well as liver size in kras+ larvae exposed to PR-39 and doxycycline were all decreased as compared to kras+ sibling controls treated with doxycycline alone and all kras− groups. Thus, there was a good correlation between numbers of infiltrated neutrophils and the size of oncogenic liver, suggesting an in vivo promoting role of neutrophils in early hepatocarcinogenesis.


Stimulation of hepatocarcinogenesis by neutrophils upon induction of oncogenic kras expression in transgenic zebrafish.

Yan C, Huo X, Wang S, Feng Y, Gong Z - J. Hepatol. (2015)

Effect of infiltrated neutrophils on liver size. (A–C) Neutrophil counts (left) and density (middle) in the liver area and liver size (right) in response to treatments of LSP (A), FPR A14 (B) or PR39 (C). Both kras−/lyz+ and kras+/lyz+ groups were similarly exposed to doxycycline from 3 dpf and neutrophils mediators were added from 4 dpf. Neutrophils and liver sizes were determined at 8 dpf (n >15 from each group). Statistical significance: *p <0.05.
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Related In: Results  -  Collection

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f0010: Effect of infiltrated neutrophils on liver size. (A–C) Neutrophil counts (left) and density (middle) in the liver area and liver size (right) in response to treatments of LSP (A), FPR A14 (B) or PR39 (C). Both kras−/lyz+ and kras+/lyz+ groups were similarly exposed to doxycycline from 3 dpf and neutrophils mediators were added from 4 dpf. Neutrophils and liver sizes were determined at 8 dpf (n >15 from each group). Statistical significance: *p <0.05.
Mentions: To further demonstrate the role of inflammatory cells in initiation and progression of hapatocarcinogenesis, we first tested a general inflammatory stimulator, LPS, which has been demonstrated to stimulate the immune system in zebrafish larvae [26]. 5 ng/ml of LPS was used to treat zebrafish larvae from 4 dpf to 8 dpf. kras+ larvae exposed to both LPS and doxycycline showed significant increases of both neutrophil count and density in the liver as compared to kras+ larvae exposed to doxycycline alone and all kras− groups. Interestingly, there was also a further enlargement of liver size with the increased neutrophils (Fig. 2A). To investigate if the accelerated liver enlargement was indeed associated with increased neutrophil infiltration, FPR-A14, which is a formyl peptide receptor agonist and has been reported to potently activate neutrophils specifically in vitro[27], was used to challenge the kras+ larvae from 4 dpf to 8 dpf. Liver neutrophil count and density in FPR-A14 and doxycycline double-treated kras+ larvae were also significantly higher than those of their kras+ sibling treated with only doxycycline and of all kras− groups (Fig. 2B), similar to that observed following LPS treatment. A further liver enlargement was also observed from these double-treated kras+ larvae. To further demonstrate the effect of neutrophils, kras+ transgenic larvae were also challenged with a neutrophil inhibitor, PR-39, a proline-rich anti-bacteria peptide that inhibits NADPH oxidase activity in neutrophils [28]. As shown in Fig. 2C, liver neutrophil count and density as well as liver size in kras+ larvae exposed to PR-39 and doxycycline were all decreased as compared to kras+ sibling controls treated with doxycycline alone and all kras− groups. Thus, there was a good correlation between numbers of infiltrated neutrophils and the size of oncogenic liver, suggesting an in vivo promoting role of neutrophils in early hepatocarcinogenesis.

Bottom Line: Both oncogenic hepatocytes and tumor-associated neutrophils (TANs) were isolated via fluorescence-activated cell sorting.Molecular analyses indicated a pro-inflammatory microenvironment, as marked by increased tgfβ1a expression in kras(V12)-expressing hepatocytes and a loss of anti-tumor activities in TANs.Depletion of Tgf-β significantly reduced the number of TANs and the size of oncogenic liver.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore; National University of Singapore Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore.

No MeSH data available.


Related in: MedlinePlus