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Toll-like receptor 3 expressing tumor parenchyma and infiltrating natural killer cells in hepatocellular carcinoma patients.

Chew V, Tow C, Huang C, Bard-Chapeau E, Copeland NG, Jenkins NA, Weber A, Lim KH, Toh HC, Heikenwalder M, Ng IO, Nardin A, Abastado JP - J. Natl. Cancer Inst. (2012)

Bottom Line: Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth.TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002).TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Singapore Immunology Network SIgN, Agency for Science, Technology and Research A*STAR, 8A Biomedical Grove, Immunos, Biopolis, Singapore 138648, Singapore.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) is a highly aggressive cancer that is linked to chronically dysregulated liver inflammation. However, appropriate immune responses can control HCC progression. Here we investigated the role and underlying mechanism of toll-like receptor 3 (TLR3) in HCC.

Methods: HCC cell death, and natural killer (NK) cell activation and cytotoxicity were assessed in vitro after treatment with the TLR3 ligand poly(I:C). The effect of TLR3 on the tumor parenchyma and infiltrating immune cells was investigated in a spontaneous liver tumor mouse model and a transplanted tumor mouse model (n = 3-9 mice per group). Immunohistochemistry and quantitative polymerase chain reaction were used to analyze tumor samples from 172 HCC patients. Paired t-tests and analysis of variance tests were used to calculate P-values. The relationship between TLR3 expression and survival was determined by the Kaplan-Meier univariate survival analysis and a log-rank test. All statistical tests were two-sided.

Results: TLR3 activation increased cell death in the TLR3(+) SNU182 HCC cell line (30.5% vs 8.5%, P = .03) and promoted NK-cell activation (32.6% vs 19.4%, P < .001) and cytotoxicity (relative fourfold increase, P = .03) in vitro. In vivo, poly(I:C) treatment increased intratumoral chemokine expression, NK-cell activation and tumor infiltration, and proliferation of tumor-infiltrating T and NK cells. Proliferation of tumor parenchyma cells was decreased. Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth. TLR3 expression in patient samples correlated with NK-cell activation, NK- and T-cell tumor infiltration, and inversely correlated with tumor parenchyma cell viability. TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002).

Conclusions: TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.

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The effect of toll-like receptor 3 (TLR3) activation by poly(I:C) on hepatocellular carcinoma cell line death. A) Dose-dependent induction of cell death in SNU182 cells was measured as Annexin V+ Topro 3+ cells by flow cytometry 24 hours after treatment with 0–100 µg/mL poly(I:C). The graph shows the means and SD from three independent experiments. A one-way analysis of variance test with Tukey’s multiple comparison post test was used to calculate two-sided P-values. In the right panel, representative dot plots of cell death 24 hours post-treatment with 50 µg/mL poly(I:C) are shown. B) Poly(I:C)-induced cell death (Annexin V+ Topro 3+ cells by flow cytometry) in SNU182 cells with TLR3 or scrambled siRNA knockdown was measured 24 hours after treatment with 50 µg/mL poly(I:C). The graphs show the means and SD from three independent experiments. Unpaired t-test was used to calculate two-sided P-values. The right panel shows representative dot plots of poly(I:C)-induced cell death with TLR3 knockdown. Scr = scrambled siRNA.
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Figure 2: The effect of toll-like receptor 3 (TLR3) activation by poly(I:C) on hepatocellular carcinoma cell line death. A) Dose-dependent induction of cell death in SNU182 cells was measured as Annexin V+ Topro 3+ cells by flow cytometry 24 hours after treatment with 0–100 µg/mL poly(I:C). The graph shows the means and SD from three independent experiments. A one-way analysis of variance test with Tukey’s multiple comparison post test was used to calculate two-sided P-values. In the right panel, representative dot plots of cell death 24 hours post-treatment with 50 µg/mL poly(I:C) are shown. B) Poly(I:C)-induced cell death (Annexin V+ Topro 3+ cells by flow cytometry) in SNU182 cells with TLR3 or scrambled siRNA knockdown was measured 24 hours after treatment with 50 µg/mL poly(I:C). The graphs show the means and SD from three independent experiments. Unpaired t-test was used to calculate two-sided P-values. The right panel shows representative dot plots of poly(I:C)-induced cell death with TLR3 knockdown. Scr = scrambled siRNA.

Mentions: To study the effects of endogenous expression of TLR3 in HCC, we screened 11 human HCC cell lines for TLR3 RNA expression by qPCR and identified seven—SNU182, SNU387, SNU449, SNU475, SK-HEP-1, HepG2, and Hep3B—that expressed the receptor (data not shown). Treatment of SNU182 cells, which exhibited high expression of TLR3, with increasing concentration of poly(I:C) for 24 hours at 37°C, resulted in a dose-dependent increase in cell death (no treatment vs 50 µg/mL poly(I:C): 8.5% vs 30.5%, P = .03; no treatment vs 100 µg/mL poly(I:C): 8.5% vs 30.9%, P = .03) (Figure 2, A). Similar results were observed for SNU449 and SNU475 cells that express moderate levels of TLR3 (Supplementary Figure 2, A, available online). TLR3 expression was increased in a dose-dependent manner by treatment with poly(I:C) in SNU182 cells (Supplementary Figure 2, B, available online). Ligands for other TLRs (lipopolysaccharide and CpG) did not induce cell death in SNU182 cells (Supplementary Figure 2, C, available online), and no effect was observed in PLC/PR5 cells that do not express TLR3 (Supplementary Figure 2, D, available online). In addition, knock-down of TLR3 in SNU182 cells reduced poly(I:C)-induced cell death (scrambled vs TLR3 siRNA: 39.9% vs 27.2%, P = .03) (Figure 2, B, and Supplementary Figure 2, E, available online). As poly(I:C) is a ligand for both TLR3 and MDA5 (21), we treated the cells with the TLR3-specific ligand poly(A:U) and observed similar results (Supplementary Figure 2, F, available online). These data indicate that TLR3 activation induces cell death in HCC cells that express endogenous TLR3.


Toll-like receptor 3 expressing tumor parenchyma and infiltrating natural killer cells in hepatocellular carcinoma patients.

Chew V, Tow C, Huang C, Bard-Chapeau E, Copeland NG, Jenkins NA, Weber A, Lim KH, Toh HC, Heikenwalder M, Ng IO, Nardin A, Abastado JP - J. Natl. Cancer Inst. (2012)

The effect of toll-like receptor 3 (TLR3) activation by poly(I:C) on hepatocellular carcinoma cell line death. A) Dose-dependent induction of cell death in SNU182 cells was measured as Annexin V+ Topro 3+ cells by flow cytometry 24 hours after treatment with 0–100 µg/mL poly(I:C). The graph shows the means and SD from three independent experiments. A one-way analysis of variance test with Tukey’s multiple comparison post test was used to calculate two-sided P-values. In the right panel, representative dot plots of cell death 24 hours post-treatment with 50 µg/mL poly(I:C) are shown. B) Poly(I:C)-induced cell death (Annexin V+ Topro 3+ cells by flow cytometry) in SNU182 cells with TLR3 or scrambled siRNA knockdown was measured 24 hours after treatment with 50 µg/mL poly(I:C). The graphs show the means and SD from three independent experiments. Unpaired t-test was used to calculate two-sided P-values. The right panel shows representative dot plots of poly(I:C)-induced cell death with TLR3 knockdown. Scr = scrambled siRNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: The effect of toll-like receptor 3 (TLR3) activation by poly(I:C) on hepatocellular carcinoma cell line death. A) Dose-dependent induction of cell death in SNU182 cells was measured as Annexin V+ Topro 3+ cells by flow cytometry 24 hours after treatment with 0–100 µg/mL poly(I:C). The graph shows the means and SD from three independent experiments. A one-way analysis of variance test with Tukey’s multiple comparison post test was used to calculate two-sided P-values. In the right panel, representative dot plots of cell death 24 hours post-treatment with 50 µg/mL poly(I:C) are shown. B) Poly(I:C)-induced cell death (Annexin V+ Topro 3+ cells by flow cytometry) in SNU182 cells with TLR3 or scrambled siRNA knockdown was measured 24 hours after treatment with 50 µg/mL poly(I:C). The graphs show the means and SD from three independent experiments. Unpaired t-test was used to calculate two-sided P-values. The right panel shows representative dot plots of poly(I:C)-induced cell death with TLR3 knockdown. Scr = scrambled siRNA.
Mentions: To study the effects of endogenous expression of TLR3 in HCC, we screened 11 human HCC cell lines for TLR3 RNA expression by qPCR and identified seven—SNU182, SNU387, SNU449, SNU475, SK-HEP-1, HepG2, and Hep3B—that expressed the receptor (data not shown). Treatment of SNU182 cells, which exhibited high expression of TLR3, with increasing concentration of poly(I:C) for 24 hours at 37°C, resulted in a dose-dependent increase in cell death (no treatment vs 50 µg/mL poly(I:C): 8.5% vs 30.5%, P = .03; no treatment vs 100 µg/mL poly(I:C): 8.5% vs 30.9%, P = .03) (Figure 2, A). Similar results were observed for SNU449 and SNU475 cells that express moderate levels of TLR3 (Supplementary Figure 2, A, available online). TLR3 expression was increased in a dose-dependent manner by treatment with poly(I:C) in SNU182 cells (Supplementary Figure 2, B, available online). Ligands for other TLRs (lipopolysaccharide and CpG) did not induce cell death in SNU182 cells (Supplementary Figure 2, C, available online), and no effect was observed in PLC/PR5 cells that do not express TLR3 (Supplementary Figure 2, D, available online). In addition, knock-down of TLR3 in SNU182 cells reduced poly(I:C)-induced cell death (scrambled vs TLR3 siRNA: 39.9% vs 27.2%, P = .03) (Figure 2, B, and Supplementary Figure 2, E, available online). As poly(I:C) is a ligand for both TLR3 and MDA5 (21), we treated the cells with the TLR3-specific ligand poly(A:U) and observed similar results (Supplementary Figure 2, F, available online). These data indicate that TLR3 activation induces cell death in HCC cells that express endogenous TLR3.

Bottom Line: Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth.TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002).TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Singapore Immunology Network SIgN, Agency for Science, Technology and Research A*STAR, 8A Biomedical Grove, Immunos, Biopolis, Singapore 138648, Singapore.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) is a highly aggressive cancer that is linked to chronically dysregulated liver inflammation. However, appropriate immune responses can control HCC progression. Here we investigated the role and underlying mechanism of toll-like receptor 3 (TLR3) in HCC.

Methods: HCC cell death, and natural killer (NK) cell activation and cytotoxicity were assessed in vitro after treatment with the TLR3 ligand poly(I:C). The effect of TLR3 on the tumor parenchyma and infiltrating immune cells was investigated in a spontaneous liver tumor mouse model and a transplanted tumor mouse model (n = 3-9 mice per group). Immunohistochemistry and quantitative polymerase chain reaction were used to analyze tumor samples from 172 HCC patients. Paired t-tests and analysis of variance tests were used to calculate P-values. The relationship between TLR3 expression and survival was determined by the Kaplan-Meier univariate survival analysis and a log-rank test. All statistical tests were two-sided.

Results: TLR3 activation increased cell death in the TLR3(+) SNU182 HCC cell line (30.5% vs 8.5%, P = .03) and promoted NK-cell activation (32.6% vs 19.4%, P < .001) and cytotoxicity (relative fourfold increase, P = .03) in vitro. In vivo, poly(I:C) treatment increased intratumoral chemokine expression, NK-cell activation and tumor infiltration, and proliferation of tumor-infiltrating T and NK cells. Proliferation of tumor parenchyma cells was decreased. Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth. TLR3 expression in patient samples correlated with NK-cell activation, NK- and T-cell tumor infiltration, and inversely correlated with tumor parenchyma cell viability. TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002).

Conclusions: TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.

Show MeSH
Related in: MedlinePlus