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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 correlation between toll-like receptor (TLR3) expression and tumor infiltration by natural killer (NK) cells and antitumor activity in hepatocellular carcinoma patients. A) The density of TLR3+ cells correlates with the density of CD56+ NK and CD8+ T cells in patient samples as determined by immunohistochemistry (n = 31). B) The density of TLR3+ cells correlates with RNA expression of interferon-γ (IFNG, n = 35) and with the density of granzyme-B (GZB)-positive cells by immunohistochemistry (n = 40) in hepatocellular carcinoma patient samples. C) The density of TLR3+ tumor parenchyma and tumor-infiltrating NK cells correlates positively with the density of Ki-67+ tumor parenchyma cells, and negatively with the density of activated caspase 3+ tumor parenchyma cells (n = 30). The Pearson correlation test was used to calculate the correlation coefficient (r) and two-sided P-values.
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Figure 6: The correlation between toll-like receptor (TLR3) expression and tumor infiltration by natural killer (NK) cells and antitumor activity in hepatocellular carcinoma patients. A) The density of TLR3+ cells correlates with the density of CD56+ NK and CD8+ T cells in patient samples as determined by immunohistochemistry (n = 31). B) The density of TLR3+ cells correlates with RNA expression of interferon-γ (IFNG, n = 35) and with the density of granzyme-B (GZB)-positive cells by immunohistochemistry (n = 40) in hepatocellular carcinoma patient samples. C) The density of TLR3+ tumor parenchyma and tumor-infiltrating NK cells correlates positively with the density of Ki-67+ tumor parenchyma cells, and negatively with the density of activated caspase 3+ tumor parenchyma cells (n = 30). The Pearson correlation test was used to calculate the correlation coefficient (r) and two-sided P-values.

Mentions: In tumor tissue from HCC patients, a higher density of TLR3-expressing cells correlated with increased tumor infiltration by CD56+ NK cells (Pearson’s r = 0.7, P < .001) and CD8+ T cells (Pearson’s r = 0.6, P <.001) (Figure 6, A). This is consistent with our previous finding that TLR3 RNA expression is associated with expression of chemokines able to attract NK cells and T cells (20). Furthermore, TLR3 protein expression correlated with NK-cell activation marker IFNG expression when measured by qPCR (Pearson’s r = 0.5, P = .001), and a similar, albeit weaker, correlation was observed with granzyme-B expression when measured by IHC (Pearson’s r = 0.4, P = .03) (Figure 6, B). Correlations between TLR3 and NCR3, CD8A, and IFNG (NK- and T-cell marker) were also observed at the RNA level in patient samples (n = 154–164) (Supplementary Table 4, available online). In addition, the density of both TLR3+ tumor parenchyma and NK cells correlated positively with tumor cell apoptosis (TLR3+ tumor parenchyma, Pearson’s r = 0.5, P = .006; TLR3+ NK cells, Pearson’s r = 0.6, P = .002) and negatively with tumor cell proliferation (TLR3+ tumor parenchyma, Pearson’s r = −0.4, P = .03; TLR3+ NK cells, Pearson’s r = −0.5, P = .005) (Figure 6, C) and were decreased in samples from patients with stage III and IV advanced HCC (Supplementary Figure 6, available online). These data from HCC patients are consistent with our in vitro and in vivo findings that TLR3 activation is associated with intratumor expression of chemokines, NK- and T-cell infiltration, NK-cell activation, and antitumor activity, as well as enhanced apoptosis and reduced proliferation of tumor parenchyma cells.


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 correlation between toll-like receptor (TLR3) expression and tumor infiltration by natural killer (NK) cells and antitumor activity in hepatocellular carcinoma patients. A) The density of TLR3+ cells correlates with the density of CD56+ NK and CD8+ T cells in patient samples as determined by immunohistochemistry (n = 31). B) The density of TLR3+ cells correlates with RNA expression of interferon-γ (IFNG, n = 35) and with the density of granzyme-B (GZB)-positive cells by immunohistochemistry (n = 40) in hepatocellular carcinoma patient samples. C) The density of TLR3+ tumor parenchyma and tumor-infiltrating NK cells correlates positively with the density of Ki-67+ tumor parenchyma cells, and negatively with the density of activated caspase 3+ tumor parenchyma cells (n = 30). The Pearson correlation test was used to calculate the correlation coefficient (r) and two-sided P-values.
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Figure 6: The correlation between toll-like receptor (TLR3) expression and tumor infiltration by natural killer (NK) cells and antitumor activity in hepatocellular carcinoma patients. A) The density of TLR3+ cells correlates with the density of CD56+ NK and CD8+ T cells in patient samples as determined by immunohistochemistry (n = 31). B) The density of TLR3+ cells correlates with RNA expression of interferon-γ (IFNG, n = 35) and with the density of granzyme-B (GZB)-positive cells by immunohistochemistry (n = 40) in hepatocellular carcinoma patient samples. C) The density of TLR3+ tumor parenchyma and tumor-infiltrating NK cells correlates positively with the density of Ki-67+ tumor parenchyma cells, and negatively with the density of activated caspase 3+ tumor parenchyma cells (n = 30). The Pearson correlation test was used to calculate the correlation coefficient (r) and two-sided P-values.
Mentions: In tumor tissue from HCC patients, a higher density of TLR3-expressing cells correlated with increased tumor infiltration by CD56+ NK cells (Pearson’s r = 0.7, P < .001) and CD8+ T cells (Pearson’s r = 0.6, P <.001) (Figure 6, A). This is consistent with our previous finding that TLR3 RNA expression is associated with expression of chemokines able to attract NK cells and T cells (20). Furthermore, TLR3 protein expression correlated with NK-cell activation marker IFNG expression when measured by qPCR (Pearson’s r = 0.5, P = .001), and a similar, albeit weaker, correlation was observed with granzyme-B expression when measured by IHC (Pearson’s r = 0.4, P = .03) (Figure 6, B). Correlations between TLR3 and NCR3, CD8A, and IFNG (NK- and T-cell marker) were also observed at the RNA level in patient samples (n = 154–164) (Supplementary Table 4, available online). In addition, the density of both TLR3+ tumor parenchyma and NK cells correlated positively with tumor cell apoptosis (TLR3+ tumor parenchyma, Pearson’s r = 0.5, P = .006; TLR3+ NK cells, Pearson’s r = 0.6, P = .002) and negatively with tumor cell proliferation (TLR3+ tumor parenchyma, Pearson’s r = −0.4, P = .03; TLR3+ NK cells, Pearson’s r = −0.5, P = .005) (Figure 6, C) and were decreased in samples from patients with stage III and IV advanced HCC (Supplementary Figure 6, available online). These data from HCC patients are consistent with our in vitro and in vivo findings that TLR3 activation is associated with intratumor expression of chemokines, NK- and T-cell infiltration, NK-cell activation, and antitumor activity, as well as enhanced apoptosis and reduced proliferation of tumor parenchyma cells.

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