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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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Related in: MedlinePlus

The association of intratumor toll-like receptor 3 (TLR3) expression by tumor parenchyma and natural killer (NK) cells with patient survival. Kaplan–Meier analyses were done to investigate the association between A) TLR3 gene expression (by quantitative polymerase chain reaction, n = 172) and B) TLR3 protein levels (by immunohistochemistry [IHC], n = 40, median = 33 cells per field) and patient survival. Log-rank Mantel–Cox test was used to calculate two-sided P-values. Representative IHC images at ×400 magnification of C) TLR3+ tumor parenchyma (red indicates TLR3 staining and blue indicates CD3 staining) and D) TLR3+ NK cells (red indicates TLR3 staining; NK cells are identified by granzyme-B expression in blue; and costaining appears purple) are shown. The insets show magnified areas. Scale bar = 30 µm. E) Kaplan–Meier analyses of TLR3+ tumor cell (median = 21 cells per field) and tumor-infiltrating NK cells densities (median = 13 cells per field) and patient survival (n = 40). The log-rank Mantel–Cox test was used to calculate two-sided P-values. CI = confidence interval, HR = hazard ratio.
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Figure 1: The association of intratumor toll-like receptor 3 (TLR3) expression by tumor parenchyma and natural killer (NK) cells with patient survival. Kaplan–Meier analyses were done to investigate the association between A) TLR3 gene expression (by quantitative polymerase chain reaction, n = 172) and B) TLR3 protein levels (by immunohistochemistry [IHC], n = 40, median = 33 cells per field) and patient survival. Log-rank Mantel–Cox test was used to calculate two-sided P-values. Representative IHC images at ×400 magnification of C) TLR3+ tumor parenchyma (red indicates TLR3 staining and blue indicates CD3 staining) and D) TLR3+ NK cells (red indicates TLR3 staining; NK cells are identified by granzyme-B expression in blue; and costaining appears purple) are shown. The insets show magnified areas. Scale bar = 30 µm. E) Kaplan–Meier analyses of TLR3+ tumor cell (median = 21 cells per field) and tumor-infiltrating NK cells densities (median = 13 cells per field) and patient survival (n = 40). The log-rank Mantel–Cox test was used to calculate two-sided P-values. CI = confidence interval, HR = hazard ratio.

Mentions: We previously reported that TLR3 gene expression in tumors was associated with overall survival among a cohort of 61 patients from Singapore (4). Here, we extended our previous study to include 111 additional patients from Hong Kong and Zurich. First, we performed qPCR on patient samples and confirmed that expression of TLR3 is associated with overall survival (HR of survival = 2.1, 95% CI = 1.3 to 3.4, P = .002) (Figure 1, A). We also observed a statistically significant association using IHC to analyze TLR3 protein expression in 40 representative tumor samples (HR of survival = 17.3, 95% CI = 6.4 to 47.1, P < .001) (Figure 1, B). TLR3 was either expressed by tumor parenchyma (Figure 1, C) or small round immune cells infiltrating the tumor nest (Figure 1, D). TLR3 was coexpressed with granzyme B but not with CD3 or CD20, suggesting that the cells that stained positively for TLR3 were NK cells (Figure 1, D) and not T or B cells (Supplementary Figure 1, available online). We next quantified TLR3 expression separately in tumor parenchyma (hepatocytes) and NK cells to determine if these two cell populations were associated with overall survival. As shown in Figure 1, E, both TLR3-expressing cell populations were associated with overall survival in Kaplan–Meier analyses (tumor parenchyma: HR of survival = 7.3, 95% CI = 2.8 to 19.1, P < .001; tumor-infiltrating NK cells: HR of survival = 8.7, 95% CI = 3.2 to 23.4, P < .001).


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 association of intratumor toll-like receptor 3 (TLR3) expression by tumor parenchyma and natural killer (NK) cells with patient survival. Kaplan–Meier analyses were done to investigate the association between A) TLR3 gene expression (by quantitative polymerase chain reaction, n = 172) and B) TLR3 protein levels (by immunohistochemistry [IHC], n = 40, median = 33 cells per field) and patient survival. Log-rank Mantel–Cox test was used to calculate two-sided P-values. Representative IHC images at ×400 magnification of C) TLR3+ tumor parenchyma (red indicates TLR3 staining and blue indicates CD3 staining) and D) TLR3+ NK cells (red indicates TLR3 staining; NK cells are identified by granzyme-B expression in blue; and costaining appears purple) are shown. The insets show magnified areas. Scale bar = 30 µm. E) Kaplan–Meier analyses of TLR3+ tumor cell (median = 21 cells per field) and tumor-infiltrating NK cells densities (median = 13 cells per field) and patient survival (n = 40). The log-rank Mantel–Cox test was used to calculate two-sided P-values. CI = confidence interval, HR = hazard ratio.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3814220&req=5

Figure 1: The association of intratumor toll-like receptor 3 (TLR3) expression by tumor parenchyma and natural killer (NK) cells with patient survival. Kaplan–Meier analyses were done to investigate the association between A) TLR3 gene expression (by quantitative polymerase chain reaction, n = 172) and B) TLR3 protein levels (by immunohistochemistry [IHC], n = 40, median = 33 cells per field) and patient survival. Log-rank Mantel–Cox test was used to calculate two-sided P-values. Representative IHC images at ×400 magnification of C) TLR3+ tumor parenchyma (red indicates TLR3 staining and blue indicates CD3 staining) and D) TLR3+ NK cells (red indicates TLR3 staining; NK cells are identified by granzyme-B expression in blue; and costaining appears purple) are shown. The insets show magnified areas. Scale bar = 30 µm. E) Kaplan–Meier analyses of TLR3+ tumor cell (median = 21 cells per field) and tumor-infiltrating NK cells densities (median = 13 cells per field) and patient survival (n = 40). The log-rank Mantel–Cox test was used to calculate two-sided P-values. CI = confidence interval, HR = hazard ratio.
Mentions: We previously reported that TLR3 gene expression in tumors was associated with overall survival among a cohort of 61 patients from Singapore (4). Here, we extended our previous study to include 111 additional patients from Hong Kong and Zurich. First, we performed qPCR on patient samples and confirmed that expression of TLR3 is associated with overall survival (HR of survival = 2.1, 95% CI = 1.3 to 3.4, P = .002) (Figure 1, A). We also observed a statistically significant association using IHC to analyze TLR3 protein expression in 40 representative tumor samples (HR of survival = 17.3, 95% CI = 6.4 to 47.1, P < .001) (Figure 1, B). TLR3 was either expressed by tumor parenchyma (Figure 1, C) or small round immune cells infiltrating the tumor nest (Figure 1, D). TLR3 was coexpressed with granzyme B but not with CD3 or CD20, suggesting that the cells that stained positively for TLR3 were NK cells (Figure 1, D) and not T or B cells (Supplementary Figure 1, available online). We next quantified TLR3 expression separately in tumor parenchyma (hepatocytes) and NK cells to determine if these two cell populations were associated with overall survival. As shown in Figure 1, E, both TLR3-expressing cell populations were associated with overall survival in Kaplan–Meier analyses (tumor parenchyma: HR of survival = 7.3, 95% CI = 2.8 to 19.1, P < .001; tumor-infiltrating NK cells: HR of survival = 8.7, 95% CI = 3.2 to 23.4, P < .001).

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