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

Valerie Chew, Charlene Tow, Caleb Huang, Emilie Bard-Chapeau, Neal G Copeland, Nancy A Jenkins, Achim Weber, Kiat Hon Lim, Han Chong Toh, Mathias Heikenwalder, Irene Oi-Lin Ng, Alessandra Nardin, Jean-Pierre Abastado, Valerie Chew, Charlene Tow, Caleb Huang, Emilie Bard-Chapeau, Neal G Copeland, Nancy A Jenkins, Achim Weber, Kiat Hon Lim, Han Chong Toh, Mathias Heikenwalder, Irene Oi-Lin Ng, Alessandra Nardin, Jean-Pierre Abastado

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.

Figures

Figure 1.
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.
Figure 2.
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.
Figure 3.
Figure 3.
The effect of toll-like receptor 3 (TLR3) activation on natural killer (NK)-cell activation and cytotoxicity against hepatocellular carcinoma cells in vitro. A) The percentage of activated NK cells coexpressing CD56 with the early activation marker CD69 was measured by flow cytometry 24 hours after treatment with 50 µg/mL poly(I:C). Data are shown using Tukey box-and-whiskers representation from 15 independent experiments. A paired t-test was used to calculate the two-sided P-value. B) Expression of the effector genes interferon-γ (IFNG) and granzyme-B (GZB) was determined by quantitative polymerase chain reaction 24 hours after NK cells were treated with 50 µg/mL poly(I:C). The graph shows the mean fold change relative to the untreated control from 12 independent experiments. Whisker bars indicate the SD. A Wilcoxon signed-rank test was used to calculate the two-sided P-values. C) The mean interferon-γ (IFN-γ) secretion by NK cells 24 hours after treatment with 50 µg/mL poly(I:C) was measured by enzyme-linked immunosorbent assay of the culture supernatant. The data are shown using Tukey box-and-whiskers representation from eight independent experiments. A paired t-test was used to calculate the two-sided P-value. D) Cytotoxicity of poly(I:C)-activated NK cells at various ratios of effector (E) to target cells (T, SNU182 cells) was measured by determining the percentage of specific lysis in vitro. A paired t-test was used to calculate the two-sided P-value at E:T ratio of 5:1. The graph shows the means from poly(I:C)-treated and untreated NK cells and the SD (whisker bars) from three independent experiments.
Figure 4.
Figure 4.
The effect of toll-like receptor 3 (TLR3) activation on intratumor chemokine expression and natural killer (NK)-cell infiltration in hepatocellular carcinoma in vivo. A) The mean RNA expression of the Ccl5 and Cxcl9 chemokines in liver tumors from mice treated with poly(I:C) (n = 6), or phosphate buffered saline (PBS)-treated mice (n = 4), or livers from non-tumor-bearing littermates (n = 2), is shown. Whisker bars indicate the SD. A one-way analysis of variance test with Bonferroni correction was used to calculate the two-sided P-values. B) In the left panel, the mean percentage of NK1.1+ CD45+ NK-cell infiltration of liver tumors harvested from poly(I:C)-treated mice (n = 6), or PBS-treated mice (n = 4), or livers from non-tumor-bearing littermates (n = 2), is shown. Whisker bars indicate the SD. A one-way analysis of variance test with Bonferroni correction was used to calculate the two-sided P-values. In the right panel, representative dot plots showing the percentage of NK1.1+ CD45+ NK cells (box) infiltrating the liver tumors harvested from poly(I:C)- and PBS-treated mice. C) The mean expression of the NK-cell effector genes perforin (Prf) and granzyme-B (Gzmb) was measured in liver tumors harvested from poly(I:C)- and PBS-treated mice using quantitative polymerase chain reaction. Graphs show means and SD. The Mann–Whitney test was used to calculate the two-sided P-values. D) Transplanted tumor growth was measured after NK cells were depleted using PK136 antibody or isotype control antibody (n = 3 per group). The graphs show the means and SD (whisker bars). A two-way analysis of variance test with Bonferroni correction was used to calculate two-sided P-values. The mean tumor area was statistically significantly different between PK136-treated and isotype control–treated mice at days 7, 10, and 14 (P = .005, <.001, and <.001, respectively).
Figure 5.
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.
Figure 6.
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.

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