Kinase inhibitor Sorafenib modulates immunosuppressive cell populations in a murine liver cancer model

Abstract

Accumulating evidence suggests that regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) are elevated in cancer patients and tumor-bearing hosts, and that depletion of Tregs and MDSC may enhance the anti-tumor immunity of the host. Sorafenib, a novel multi-kinase inhibitor, is approved for the treatment of several human cancers, including advanced hepatocellular carcinoma (HCC). Sorafenib is believed to inhibit tumor growth via anti-angiogenesis, cell cycle arrest, and inducing apoptosis. However, the impact of Sorafenib on immune cell populations in tumor-bearing hosts is unclear. In this report, we show that Tregs and MDSC are increased in the spleens and bone marrows of the BALB/c mice with liver hepatoma. The increase in Tregs and MDSC was positively correlated with tumor burden. Treatment of Sorafenib not only inhibited HCC cell growth in mice but also significantly decreased the suppressive immune cell populations: Tregs and MDSC. In conclusion, our study strongly suggests that Sorafenib can enhance anti-tumor immunity via modulating immunosuppressive cell populations in the murine liver cancer model.

Figure 1. Tumor growth and enlarged spleens in immune competent BALB/c mice

Six to eight weeks old BALB/c mice were injected (s.c.) 1MEA cells ( 5 × 10⁶ / per mouse). After two weeks, the tumors were measurable. As mentioned in Materials and Methods, at the endpoint of experiments (the end of the forth week), spleens and tumor tissues were collected and weighed. Splenocytes were prepared and counted after red cells lysis. A. Tumor-bearing BALB/c mice; B. Tumor tissue separated from the mice. C. Histological features of the liver cancer tissue (H&E stain) showing poorly differentiated hepatocellular carcinoma; D. the size of spleens from Balb/c mice with tumor (right) and normal mice (left); E. the weight of spleens from normal mice and tumor-bearing mice (n=9); F. the splenocytes from normal mice (87± 14 × 10⁶; n=3) and tumor-bearing mice (263 ± 95 × 10⁶; n=5). Data represent the mean ± SD from one of two similar experiments

Figure 2. Increased CD4⁺CD25⁺ T cells and CD11b⁺Gr-1⁺ cells in tumor-bearing mice

A. A representative flow cytometric data showing the CD4⁺CD25⁺ T cells frequency of gated CD4⁺ T cells in splenocytes from normal and HCC tumor-bearing mice; B. Mean CD4⁺CD25⁺ T cells frequency in CD4⁺ T cells in splenocytes from normal and HCC tumor-bearing mice (n=3) . C and D. A representative (C) and mean ( D) flow cytometry data showing increased CD11b⁺ Gr-1⁺cells frequency in splenocytes from normal and HCC tumor-bearing mice (n=3). The gated viable cells were analyzed. Numbers in the figures represent the percentage of fluorescence-positive cells in corresponding areas; E. Functional analysis of CD11b⁺ cells were evaluated for their ability to suppress autologous CD11b⁺-depleted splenocytes proliferation by [³H]-thymidine incorporation when stimulated with anti-CD3 and CD28 at various ratios. Values represent mean ± SD (n=3) and are representative examples of three separate experiments.

Figure 2. Increased CD4⁺CD25⁺ T cells and CD11b⁺Gr-1⁺ cells in tumor-bearing mice

A. A representative flow cytometric data showing the CD4⁺CD25⁺ T cells frequency of gated CD4⁺ T cells in splenocytes from normal and HCC tumor-bearing mice; B. Mean CD4⁺CD25⁺ T cells frequency in CD4⁺ T cells in splenocytes from normal and HCC tumor-bearing mice (n=3) . C and D. A representative (C) and mean ( D) flow cytometry data showing increased CD11b⁺ Gr-1⁺cells frequency in splenocytes from normal and HCC tumor-bearing mice (n=3). The gated viable cells were analyzed. Numbers in the figures represent the percentage of fluorescence-positive cells in corresponding areas; E. Functional analysis of CD11b⁺ cells were evaluated for their ability to suppress autologous CD11b⁺-depleted splenocytes proliferation by [³H]-thymidine incorporation when stimulated with anti-CD3 and CD28 at various ratios. Values represent mean ± SD (n=3) and are representative examples of three separate experiments.

Figure 3. Sorafnib inhibits HCC growth in BALB/c mice

Blab/c mice of 6-8 weeks were subcutaneously injected with 1MEA cells (4 million/mouse) in the right flank. Two weeks later, the mice with measurable tumor were divided into two groups. Each mouse was gavage-fed with Sorafenib (0.6mg/100ul) or solvent (100ul), once daily, five days each week, for two weeks. At endpoint of experiments, mice were sacrificed, tumors and spleens were collected and weighed. A. A representative of HCC tumor-bearing mice after 2 weeks treatment with Soarfenib (left) or solvent (right), spleen (right upper) and tumor (right lower) size. B. the weight of tumor tissues from tumor-bearing mice after 2 weeks treatment with sorafenib or solvent (n=5). C. the weight of spleens from normal mice or tumor-bearing mice after 2 weeks treatment with sorafenib or solvent (n=5). Data represent the mean ± SD of 5 mice per group from one representative experiment of two similar experiments. * P ≤ 0.05

Figure 4. Sorafenib treatment significantly decreased the percentage of CD4⁺CD25⁺ T cells and CD11b⁺ cells in tumor-bearing mice

A and B. A representative of flow cytometry data showing the frequency of CD4⁺CD25⁺ T cells and CD11b⁺Gr-1⁺ cells in spleens and bone marrow from normal mice or tumor-bearing mice treated with solvent or sorafenib; C and D. A representative (C) and collective ( D, n=3) of flow cytometry data showing the expression of FOXP3 in CD4⁺CD25⁺ T cells in spleens from normal mice or tumor-bearing mice treated with solvent or sorafenib; E. A representative of flow cytomrtey data showing the frequency of CD11b⁺ Gr-1⁺cells in bone marrow (upper panel) and spleen (lower panel) from normal mice or tumor-bearing mice treated with solvent or sorafenib; F. Mean CD11b⁺ Gr-1⁺ cells frequency bone marrow and spleen from normal and HCC tumor-bearing mice treated with solvent or sorafenib (n=5) . Data represent the mean ± SD of 3 or 5 mice per group from one representative experiment of two similar experiments.

Figure 4. Sorafenib treatment significantly decreased the percentage of CD4⁺CD25⁺ T cells and CD11b⁺ cells in tumor-bearing mice

A and B. A representative of flow cytometry data showing the frequency of CD4⁺CD25⁺ T cells and CD11b⁺Gr-1⁺ cells in spleens and bone marrow from normal mice or tumor-bearing mice treated with solvent or sorafenib; C and D. A representative (C) and collective ( D, n=3) of flow cytometry data showing the expression of FOXP3 in CD4⁺CD25⁺ T cells in spleens from normal mice or tumor-bearing mice treated with solvent or sorafenib; E. A representative of flow cytomrtey data showing the frequency of CD11b⁺ Gr-1⁺cells in bone marrow (upper panel) and spleen (lower panel) from normal mice or tumor-bearing mice treated with solvent or sorafenib; F. Mean CD11b⁺ Gr-1⁺ cells frequency bone marrow and spleen from normal and HCC tumor-bearing mice treated with solvent or sorafenib (n=5) . Data represent the mean ± SD of 3 or 5 mice per group from one representative experiment of two similar experiments.

Figure 4. Sorafenib treatment significantly decreased the percentage of CD4⁺CD25⁺ T cells and CD11b⁺ cells in tumor-bearing mice

A and B. A representative of flow cytometry data showing the frequency of CD4⁺CD25⁺ T cells and CD11b⁺Gr-1⁺ cells in spleens and bone marrow from normal mice or tumor-bearing mice treated with solvent or sorafenib; C and D. A representative (C) and collective ( D, n=3) of flow cytometry data showing the expression of FOXP3 in CD4⁺CD25⁺ T cells in spleens from normal mice or tumor-bearing mice treated with solvent or sorafenib; E. A representative of flow cytomrtey data showing the frequency of CD11b⁺ Gr-1⁺cells in bone marrow (upper panel) and spleen (lower panel) from normal mice or tumor-bearing mice treated with solvent or sorafenib; F. Mean CD11b⁺ Gr-1⁺ cells frequency bone marrow and spleen from normal and HCC tumor-bearing mice treated with solvent or sorafenib (n=5) . Data represent the mean ± SD of 3 or 5 mice per group from one representative experiment of two similar experiments.

Figure 4. Sorafenib treatment significantly decreased the percentage of CD4⁺CD25⁺ T cells and CD11b⁺ cells in tumor-bearing mice

A and B. A representative of flow cytometry data showing the frequency of CD4⁺CD25⁺ T cells and CD11b⁺Gr-1⁺ cells in spleens and bone marrow from normal mice or tumor-bearing mice treated with solvent or sorafenib; C and D. A representative (C) and collective ( D, n=3) of flow cytometry data showing the expression of FOXP3 in CD4⁺CD25⁺ T cells in spleens from normal mice or tumor-bearing mice treated with solvent or sorafenib; E. A representative of flow cytomrtey data showing the frequency of CD11b⁺ Gr-1⁺cells in bone marrow (upper panel) and spleen (lower panel) from normal mice or tumor-bearing mice treated with solvent or sorafenib; F. Mean CD11b⁺ Gr-1⁺ cells frequency bone marrow and spleen from normal and HCC tumor-bearing mice treated with solvent or sorafenib (n=5) . Data represent the mean ± SD of 3 or 5 mice per group from one representative experiment of two similar experiments.

Figure 5. Sorafenib treatment maintained the CD4 and CD8 cells in spleen and lymph nodes

A representative flow cytometric data showing the CD4⁺and CD8⁺ T cells frequency of gated lymphocytes in spleen and lymph nodes from normal and HCC tumor-bearing mice treated with solvent or soarfenib. Numbers in the figures represent the percentage of fluorescence-positive cells in corresponding areas, representative data of three independent experiments.