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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 on antitumor activity in vivo. A) The mean number of Ki67+CD45+ proliferating tumor-infiltrating leukocytes per field was assessed by immunohistochemistry in liver tumors from poly(I:C)-treated mice compared with phosphate buffered saline (PBS)-treated mice. Whisker bars indicate the SD. An unpaired t-test was used to calculate the two-sided P-value. B) Representative immunohistochemical images of CD3 or granzyme-B (GZB) staining (red) and Ki67 (blue) at ×400 magnification. Membrane expression of CD3, cytoplasmic expression of GZB, and nuclear expression of Ki-67 were observed. The insets show a representative magnified area. Scale bar = 30 µm. C) The mean number of Ki67+CD45- proliferating tumor parenchyma cells per field in liver tumors harvested from poly(I:C)-treated mice compared with PBS-treated mice is shown. Whisker bars indicate the SD. The Mann–Whitney test was used to calculate the two-sided P-value. D) The mean number of apoptotic tumor parenchyma cells per field detected by terminal deoxynucleotidyl transferase dUTP nick end labeling in liver tumors harvested from poly(I:C)-treated or PBS-treated mice is shown. Whisker bars indicate the SD. The Mann–Whitney test was used to calculate the two-sided P-value. Representative immunohistochemical images are also shown at ×200 magnification. Scale bar = 50 µm. E) Transplanted tumor growth was determined in mice treated with poly(I:C) vs PBS (n = 6 mice per group). On day 14, the mean tumor area of poly(I:C)- vs PBS-treated mice is 14.5 vs 39.5mm2, P < .001. Whisker bars indicate the SD. A two-way analysis of variance test with Bonferroni correction was used to calculate the two-sided P-value. F) Relative gene expression of T (threefold increase in Cd3g) and NK (twofold increase in Nkp46) cell markers in transplanted tumors treated with poly(I:C) vs PBS (n = 6 mice per group) is shown. The graphs show the means and SD (whisker bars). An unpaired t-test was used to calculate the two-sided P-values.
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Figure 5: The effect of toll-like receptor 3 (TLR3) activation on antitumor activity in vivo. A) The mean number of Ki67+CD45+ proliferating tumor-infiltrating leukocytes per field was assessed by immunohistochemistry in liver tumors from poly(I:C)-treated mice compared with phosphate buffered saline (PBS)-treated mice. Whisker bars indicate the SD. An unpaired t-test was used to calculate the two-sided P-value. B) Representative immunohistochemical images of CD3 or granzyme-B (GZB) staining (red) and Ki67 (blue) at ×400 magnification. Membrane expression of CD3, cytoplasmic expression of GZB, and nuclear expression of Ki-67 were observed. The insets show a representative magnified area. Scale bar = 30 µm. C) The mean number of Ki67+CD45- proliferating tumor parenchyma cells per field in liver tumors harvested from poly(I:C)-treated mice compared with PBS-treated mice is shown. Whisker bars indicate the SD. The Mann–Whitney test was used to calculate the two-sided P-value. D) The mean number of apoptotic tumor parenchyma cells per field detected by terminal deoxynucleotidyl transferase dUTP nick end labeling in liver tumors harvested from poly(I:C)-treated or PBS-treated mice is shown. Whisker bars indicate the SD. The Mann–Whitney test was used to calculate the two-sided P-value. Representative immunohistochemical images are also shown at ×200 magnification. Scale bar = 50 µm. E) Transplanted tumor growth was determined in mice treated with poly(I:C) vs PBS (n = 6 mice per group). On day 14, the mean tumor area of poly(I:C)- vs PBS-treated mice is 14.5 vs 39.5mm2, P < .001. Whisker bars indicate the SD. A two-way analysis of variance test with Bonferroni correction was used to calculate the two-sided P-value. F) Relative gene expression of T (threefold increase in Cd3g) and NK (twofold increase in Nkp46) cell markers in transplanted tumors treated with poly(I:C) vs PBS (n = 6 mice per group) is shown. The graphs show the means and SD (whisker bars). An unpaired t-test was used to calculate the two-sided P-values.

Mentions: We next characterized the functional status of TILs in poly(I:C)-treated mice. As shown in Figure 5, A, the frequency of Ki67+CD45+ proliferating TILs was ninefold higher in liver tumors from poly(I:C)- vs PBS-treated mice (mean number of Ki67+CD45+ TILs per field = 167 vs 19, respectively, P < .001). Furthermore, double-IHC using antibodies against Ki67 with either CD3 or granzyme B identified these proliferating TILs as T cells and NK cells (Figure 5, B). Conversely, proliferation of tumor parenchyma cells (identified as Ki67+CD45− cells with distinct morphology) was fourfold lower in poly(I:C)- vs PBS-treated mice (mean = 41 vs 166 cells per field, respectively, P = .002) (Figure 5, C), whereas apoptosis of tumor cells was enhanced by 11-fold (mean = 32 vs 3 cells per field, respectively, P < .001) (Figure 5, D). Similar results were observed when mice were treated with poly(A:U) (Supplementary Figure 4, C and D, available online). These data show that TLR3 activation in tumor-bearing mice increased the proliferation of tumor-infiltrating T and NK cells whereas tumor-cell proliferation and viability were decreased.


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 on antitumor activity in vivo. A) The mean number of Ki67+CD45+ proliferating tumor-infiltrating leukocytes per field was assessed by immunohistochemistry in liver tumors from poly(I:C)-treated mice compared with phosphate buffered saline (PBS)-treated mice. Whisker bars indicate the SD. An unpaired t-test was used to calculate the two-sided P-value. B) Representative immunohistochemical images of CD3 or granzyme-B (GZB) staining (red) and Ki67 (blue) at ×400 magnification. Membrane expression of CD3, cytoplasmic expression of GZB, and nuclear expression of Ki-67 were observed. The insets show a representative magnified area. Scale bar = 30 µm. C) The mean number of Ki67+CD45- proliferating tumor parenchyma cells per field in liver tumors harvested from poly(I:C)-treated mice compared with PBS-treated mice is shown. Whisker bars indicate the SD. The Mann–Whitney test was used to calculate the two-sided P-value. D) The mean number of apoptotic tumor parenchyma cells per field detected by terminal deoxynucleotidyl transferase dUTP nick end labeling in liver tumors harvested from poly(I:C)-treated or PBS-treated mice is shown. Whisker bars indicate the SD. The Mann–Whitney test was used to calculate the two-sided P-value. Representative immunohistochemical images are also shown at ×200 magnification. Scale bar = 50 µm. E) Transplanted tumor growth was determined in mice treated with poly(I:C) vs PBS (n = 6 mice per group). On day 14, the mean tumor area of poly(I:C)- vs PBS-treated mice is 14.5 vs 39.5mm2, P < .001. Whisker bars indicate the SD. A two-way analysis of variance test with Bonferroni correction was used to calculate the two-sided P-value. F) Relative gene expression of T (threefold increase in Cd3g) and NK (twofold increase in Nkp46) cell markers in transplanted tumors treated with poly(I:C) vs PBS (n = 6 mice per group) is shown. The graphs show the means and SD (whisker bars). An unpaired t-test was used to calculate the two-sided P-values.
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Figure 5: The effect of toll-like receptor 3 (TLR3) activation on antitumor activity in vivo. A) The mean number of Ki67+CD45+ proliferating tumor-infiltrating leukocytes per field was assessed by immunohistochemistry in liver tumors from poly(I:C)-treated mice compared with phosphate buffered saline (PBS)-treated mice. Whisker bars indicate the SD. An unpaired t-test was used to calculate the two-sided P-value. B) Representative immunohistochemical images of CD3 or granzyme-B (GZB) staining (red) and Ki67 (blue) at ×400 magnification. Membrane expression of CD3, cytoplasmic expression of GZB, and nuclear expression of Ki-67 were observed. The insets show a representative magnified area. Scale bar = 30 µm. C) The mean number of Ki67+CD45- proliferating tumor parenchyma cells per field in liver tumors harvested from poly(I:C)-treated mice compared with PBS-treated mice is shown. Whisker bars indicate the SD. The Mann–Whitney test was used to calculate the two-sided P-value. D) The mean number of apoptotic tumor parenchyma cells per field detected by terminal deoxynucleotidyl transferase dUTP nick end labeling in liver tumors harvested from poly(I:C)-treated or PBS-treated mice is shown. Whisker bars indicate the SD. The Mann–Whitney test was used to calculate the two-sided P-value. Representative immunohistochemical images are also shown at ×200 magnification. Scale bar = 50 µm. E) Transplanted tumor growth was determined in mice treated with poly(I:C) vs PBS (n = 6 mice per group). On day 14, the mean tumor area of poly(I:C)- vs PBS-treated mice is 14.5 vs 39.5mm2, P < .001. Whisker bars indicate the SD. A two-way analysis of variance test with Bonferroni correction was used to calculate the two-sided P-value. F) Relative gene expression of T (threefold increase in Cd3g) and NK (twofold increase in Nkp46) cell markers in transplanted tumors treated with poly(I:C) vs PBS (n = 6 mice per group) is shown. The graphs show the means and SD (whisker bars). An unpaired t-test was used to calculate the two-sided P-values.
Mentions: We next characterized the functional status of TILs in poly(I:C)-treated mice. As shown in Figure 5, A, the frequency of Ki67+CD45+ proliferating TILs was ninefold higher in liver tumors from poly(I:C)- vs PBS-treated mice (mean number of Ki67+CD45+ TILs per field = 167 vs 19, respectively, P < .001). Furthermore, double-IHC using antibodies against Ki67 with either CD3 or granzyme B identified these proliferating TILs as T cells and NK cells (Figure 5, B). Conversely, proliferation of tumor parenchyma cells (identified as Ki67+CD45− cells with distinct morphology) was fourfold lower in poly(I:C)- vs PBS-treated mice (mean = 41 vs 166 cells per field, respectively, P = .002) (Figure 5, C), whereas apoptosis of tumor cells was enhanced by 11-fold (mean = 32 vs 3 cells per field, respectively, P < .001) (Figure 5, D). Similar results were observed when mice were treated with poly(A:U) (Supplementary Figure 4, C and D, available online). These data show that TLR3 activation in tumor-bearing mice increased the proliferation of tumor-infiltrating T and NK cells whereas tumor-cell proliferation and viability were decreased.

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