Tumor Induced Hepatic Myeloid Derived Suppressor Cells Can Cause Moderate Liver Damage

<div><p>Subcutaneous tumors induce the accumulation of myeloid derived suppressor cells (MDSC) not only in blood and spleens, but also in livers of these animals. Unexpectedly, we observed a moderate increase in serum transaminases in mice with EL4 subcutaneous tumors, which prompted us to study the relationship of hepatic MDSC accumulation and liver injury. MDSC were the predominant immune cell population expanding in livers of all subcutaneous tumor models investigated (RIL175, B16, EL4, CT26 and BNL), while liver injury was only observed in EL4 and B16 tumor-bearing mice. Elimination of hepatic MDSC in EL4 tumor-bearing mice using low dose 5-fluorouracil (5-FU) treatment reversed transaminase elevation and adoptive transfer of hepatic MDSC from B16 tumor-bearing mice caused transaminase elevation indicating a direct MDSC mediated effect. Surprisingly, hepatic MDSC from B16 tumor-bearing mice partially lost their damage-inducing potency when transferred into mice bearing non damage-inducing RIL175 tumors. Furthermore, MDSC expansion and MDSC-mediated liver injury further increased with growing tumor burden and was associated with different cytokines including GM-CSF, VEGF, interleukin-6, CCL2 and KC, depending on the tumor model used. In contrast to previous findings, which have implicated MDSC only in protection from T cell-mediated hepatitis, we show that tumor-induced hepatic MDSC themselves can cause moderate liver damage.</p></div

Cytokine secretion profiles of different tumor models.

<p>Duplicates of tumor-conditioned media (A, N = 4–6 media samples per tumor cell line culture) or serum samples from tumor-bearing mice (B, N = 4–6 serum samples per group) were analyzed for interleukin-6, CCL-2, GM-CSF, M-CSF, KC and VEGF (A) or interleukin-6, CCL-2, KC, VEGF, IFN-γ and interleukin 10 (B). Serum samples from tumor-bearing mice were normalized to serum from naïve wild-type mice. ND  =  not detected. Data are expressed as mean ±SEM. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 (by One-way ANOVA).</p

Increased expansion of liver damage-inducing MDSC exacerbates liver damage.

<p>Mice with different size subcutaneous tumors were analyzed for absolute numbers of hepatic MDSC (A and B), M-MDSC or (C), PMN-MDSC (D) and serum ALT levels (B–D). B–D, graphs correlate ALT levels with absolute numbers of MDSC and MDSC subsets. (N = 6–9 mice per tumor, 3 independent experiments). Data are expressed as mean ±SEM. *<i>p</i><0.05, **<i>p</i><0.01 (by two-tailed Student's <i>t</i> test).</p

Melanoma and lymphoma subcutaneous tumor-bearing mice suffer from mild liver damage.

<p>C57BL/6 and BALB/c mice bearing indicated subcutaneous tumors were sacrificed, when tumor diameter reached 15 mm. ALT (A) and AST (B) levels were analyzed in mouse serum (N≥8 mice per tumor, N≥6 naïve mice, 3 independent experiments). Naïve C57BL/6 mice (C, left image) or mice bearing B16 subcutaneous tumors (C, right image) were sacrificed, when tumor diameter reached 20 mm. TUNEL assays were performed on liver specimen (C; scale bar  = 100 µm; N = 2 mice per group, total of 5 TUNEL assays per group) and TUNEL positive cells were counted in 20 non-overlapping visual fields. Means of TUNEL positive cells per liver section were plotted (D). C, Representative examples of visual fields are shown. Data are expressed as mean ±SEM. *<i>p</i><0.05, ***<i>p</i><0.001, ****<i>p</i><0.0001 (by One-way ANOVA).</p

Liver injury depends on the presence of hepatic MDSC with damage-inducing potency.

<p>EL4 tumor-bearing mice were treated with 5-FU or saline. Liver immune cells were analyzed for MDSC and MDSC subsets and mouse serum was analyzed for ALT and AST levels (A) (N = 6 mice per treatment group, 2 independent experiments). B, 5×10⁷ CD11b⁺ cells isolated from livers of indicated untreated subcutaneous tumor-bearing mice were injected intravenously into naïve or RIL175 tumor-bearing recipient mice and ALT and AST serum levels were analyzed 16 h after transfer (N≥6 recipient mice, 2 independent experiments). Data are expressed as mean ±SEM. **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001 (A was analyzed by two-tailed Student's <i>t</i> test. B was analyzed by One-way ANOVA).</p

Analysis of hepatic immune cells in mice with subcutaneous tumors.

<p>C57BL/6 naïve mice or mice bearing EL4 or B16 tumors were sacrificed, when tumor diameter reached 15 mm. Hepatic immune cells were analyzed by flow cytometry and frequency and absolute cell number per gram liver were calculated for the myeloid compartment (A) and the lymphoid compartment (B) (N = 5 mice per tumor). C, Frequencies of CD11b⁺Gr-1⁺CD244⁺ cells in livers of naïve mice or mice bearing indicated tumors (N = 3 mice per group). D, Change of frequency of myeloid (including MDSC) and lymphoid cells in naïve vs. EL4 or B16 tumor-bearing mice. E, fold increase of absolute numbers of MDSC (CD11b⁺Gr-1⁺ cells) or non-MDSC (total number of liver leukocytes minus number of CD11b⁺Gr-1⁺ cells) in tumor bearing vs. naïve mice (N = 8 mice per tumor). Data are expressed as mean ±SEM. *<i>p</i><0.05, **<i>p</i><0.01 (C was analyzed by One-way ANOVA. E was analyzed by two-tailed Student's <i>t</i> test).</p