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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

Analyses of FACS-isolated hepatocytes and neutrophils. (A) FACS profiles of liver cells from kras+/lyz+, fabp10+ and kras−/lyz+ fish. Fluorescent protein-labeled hepatocytes and neutrophils are boxed in each profile. Both cell populations are boxed. (B–D) RT-qPCR determination of RNA expression of selected genes in krasV12-expressing hepatocytes (B), TANs (C) and Tgf-β signaling blocked TANs (D). Fold changes in log2 scale are shown for krasV12-expressing hepatocytes vs. krasV12 non-expressing fabp10+ hepatocytes (B), TANs vs. NNs (C) and Tgf-β signaling blocked TANs vs. control TANs (D). All quantifications were repeated in three experiments and the same trend was observed. *p <0.05. (This figure appears in colour on the web.)
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f0035: Analyses of FACS-isolated hepatocytes and neutrophils. (A) FACS profiles of liver cells from kras+/lyz+, fabp10+ and kras−/lyz+ fish. Fluorescent protein-labeled hepatocytes and neutrophils are boxed in each profile. Both cell populations are boxed. (B–D) RT-qPCR determination of RNA expression of selected genes in krasV12-expressing hepatocytes (B), TANs (C) and Tgf-β signaling blocked TANs (D). Fold changes in log2 scale are shown for krasV12-expressing hepatocytes vs. krasV12 non-expressing fabp10+ hepatocytes (B), TANs vs. NNs (C) and Tgf-β signaling blocked TANs vs. control TANs (D). All quantifications were repeated in three experiments and the same trend was observed. *p <0.05. (This figure appears in colour on the web.)

Mentions: It has been well established that cancer cells are capable of creating a pro-inflammatory microenvironment [31,32]. To investigate molecular interaction of oncogenic hepatocytes and neutrophils in our model, GFP-krasG12V-expressing hepatocytes were isolated from doxycycline-treated kras+ larvae by FACS and meanwhile enriched TANs were isolated based on DsRed expression (Fig. 7A). RT-qPCR analysis of kras+ hepatocytes (Fig. 7B) showed a significant upregulation of tgfβ1a, a primary regulator in early development of liver fibrosis and cancer-related inflammation [33,34]. In contrast, key anti-tumor genes such as tnfa and ifnγ showed significant downregulation. Meanwhile, compared to NNs, TANs showed a general pro-tumor gene expression pattern. For instance, il1b, which promotes early cancer angiogenesis [35], was significantly upregulated while anti-tumor cytokines, il4, il6, il8, il10, il12, and tnfa, all showed significant downregulation (Fig. 7C). Tgf-β is known to polarize neutrophils to a pro-tumor phenotype [4]. To investigate if oncogenic hepatocyte-secreted Tgf-β mediated crosstalk between the two cell types, SB431542, a specific inhibitor of Tgf-β type I receptor [36,37], was used to block Tgf-β signaling in TANs where both tgfβr1a and tgfβr1b were found to be expressed (data not shown). Compared to unblocked TANs, significant upregulation of all but one examined anti-tumor cytokines (il6, il8, il10, il12, and tnfa) was observed in Tgf-β receptor blocked TANs. Thus, blockage of Tgf-β signaling partially rescued the expression of these anti-tumor genes (Fig. 7D).


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)

Analyses of FACS-isolated hepatocytes and neutrophils. (A) FACS profiles of liver cells from kras+/lyz+, fabp10+ and kras−/lyz+ fish. Fluorescent protein-labeled hepatocytes and neutrophils are boxed in each profile. Both cell populations are boxed. (B–D) RT-qPCR determination of RNA expression of selected genes in krasV12-expressing hepatocytes (B), TANs (C) and Tgf-β signaling blocked TANs (D). Fold changes in log2 scale are shown for krasV12-expressing hepatocytes vs. krasV12 non-expressing fabp10+ hepatocytes (B), TANs vs. NNs (C) and Tgf-β signaling blocked TANs vs. control TANs (D). All quantifications were repeated in three experiments and the same trend was observed. *p <0.05. (This figure appears in colour on the web.)
© Copyright Policy
Related In: Results  -  Collection

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f0035: Analyses of FACS-isolated hepatocytes and neutrophils. (A) FACS profiles of liver cells from kras+/lyz+, fabp10+ and kras−/lyz+ fish. Fluorescent protein-labeled hepatocytes and neutrophils are boxed in each profile. Both cell populations are boxed. (B–D) RT-qPCR determination of RNA expression of selected genes in krasV12-expressing hepatocytes (B), TANs (C) and Tgf-β signaling blocked TANs (D). Fold changes in log2 scale are shown for krasV12-expressing hepatocytes vs. krasV12 non-expressing fabp10+ hepatocytes (B), TANs vs. NNs (C) and Tgf-β signaling blocked TANs vs. control TANs (D). All quantifications were repeated in three experiments and the same trend was observed. *p <0.05. (This figure appears in colour on the web.)
Mentions: It has been well established that cancer cells are capable of creating a pro-inflammatory microenvironment [31,32]. To investigate molecular interaction of oncogenic hepatocytes and neutrophils in our model, GFP-krasG12V-expressing hepatocytes were isolated from doxycycline-treated kras+ larvae by FACS and meanwhile enriched TANs were isolated based on DsRed expression (Fig. 7A). RT-qPCR analysis of kras+ hepatocytes (Fig. 7B) showed a significant upregulation of tgfβ1a, a primary regulator in early development of liver fibrosis and cancer-related inflammation [33,34]. In contrast, key anti-tumor genes such as tnfa and ifnγ showed significant downregulation. Meanwhile, compared to NNs, TANs showed a general pro-tumor gene expression pattern. For instance, il1b, which promotes early cancer angiogenesis [35], was significantly upregulated while anti-tumor cytokines, il4, il6, il8, il10, il12, and tnfa, all showed significant downregulation (Fig. 7C). Tgf-β is known to polarize neutrophils to a pro-tumor phenotype [4]. To investigate if oncogenic hepatocyte-secreted Tgf-β mediated crosstalk between the two cell types, SB431542, a specific inhibitor of Tgf-β type I receptor [36,37], was used to block Tgf-β signaling in TANs where both tgfβr1a and tgfβr1b were found to be expressed (data not shown). Compared to unblocked TANs, significant upregulation of all but one examined anti-tumor cytokines (il6, il8, il10, il12, and tnfa) was observed in Tgf-β receptor blocked TANs. Thus, blockage of Tgf-β signaling partially rescued the expression of these anti-tumor genes (Fig. 7D).

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