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

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

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

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

Hepatic tumor nodule incidence by ApoLinkerP144 is also diminished in immunodeficient mice by modulating TGF-β–dependent molecules.

<p>(A) 5×10⁵ MC38 colon carcinoma cells were intrasplenically injected and 4×10¹² AAVApo or AAVApoLinkerP144 vg/mice were simultaneously i.p injected in <i>Rag2^−/−IL2rγ^−/−</i> mice (<i>n</i> = 6/group). Mice were sacrificed at day 10 after the tumor cell inoculation and the tumor area in the liver was measured quantifying the pixels over a threshold color using Matlab software. Mean±SEM *, <i>P</i><0.05. (B) FGFR1, MMP9, periostin and COX-2 expression in tumor liver metastases from <i>Rag2^−/−IL2rγ^−/−</i> mice was assessed by RT-PCR. Mean±SEM *, <i>P</i><0.05.</p

Blockade of TGF-β <i>in vivo</i> by ApoLinkerP144 delivered either by gene therapy or bound to HDL.

<p>(A) Western blot against ApoA-I with purified HDL fractions of plasma obtained from animals which received 24 h before either pApo or pApoLinkerP144 by hydrodynamic injection. (B) Inhibition of TGF-β by HDL from mice injected with Apo plasmid (HDL Apo) or HDL containing ApoLinkerP144 (HDL ApoLinkerP144) measured using Mv-1-Lu cells cultured in the presence of 200 pg/ml of TGF-β and 10 µg/ml HDLs. Mean±SEM *, <i>P</i><0.05 (C) Strengthened pStat-1 expression and diminished pSmad-2 expression in livers from mice which received IL-12 plasmid and HDL Apo or HDL ApoLinkerP144. Numbers indicate the relative intensity of the bands. (D) IFN-γ production 4 days after IL-12 plasmid hydrodynamic injection combined with i.p injection of HDL Apo or HDL ApoLinkerP144 or a hydrodynamic injection of pApoLinkerP144 in C57BL/6 mice (<i>n</i> = 4 mice/group). Mean±SEM **, <i>P</i><0.01.</p

Sustained expression of ApoLinkerP144 delays tumor progression and increases intratumoral CD8 T cell infiltration in a mouse model of spontaneous melanoma.

<p>(A) Scavenger Receptor B class 1 (SRB1) expression on two different Ret cell lines (established from primary skin melanomas) analyzed by flow cytometry. (B) <i>Ret</i> transgenic tumor-bearing mice were injected i.p either with saline or 4×10¹² gc/mouse of AAVApoLinkerP144 (<i>n</i> = 7/group). Results are displayed as Kaplan-Meier plot of mice survival. Cumulative data of two independent experiments are shown. (C) <i>Ret</i> transgenic mice were injected i.p either with saline or 4×10¹² gc/mouse of AAVApoLinkerP144. Then, mice were sacrificed either at day 21 (<i>n</i> = 7/group) or 50 (saline, <i>n</i> = 5; treatment <i>n</i> = 11) after injection. Mice sacrificed at day 50 were classified as responder (R; grey bar, <i>n</i> = 6) or non-responder (NR; white bar, <i>n</i> = 5) regarding tumor weight; (D) Cells from primary skin tumors and metastatic lymph nodes were isolated at day 50 upon the treatment Frequency of CD8⁺ T lymphocytes in metastatic lymph nodes and of tumor-infiltrating dendritic cells producing TNF-α upon the treatment overnight with LPS measured by flow cytometry were presented as a percentage within the respective cell population; Mean±SEM *, <i>P</i><0.05.</p

Sustained expression of ApoLinkerP144 in the liver using an AAV vector significantly diminishes the occurrence of primary liver metastasis.

<p>(A) ApoA-I Western blot analysis performed on supernatants of 293T cells producing the AAV vectors. (B) ApoA-I deficient mice received 4×10¹¹ genome copies/mice i.v. (<i>n</i> = 6/group) and ApoA-I production in serum was quantified by ELISA. (C) 5×10⁵ MC38 colon carcinoma cells were intrasplenically injected. At the same time, 4×10¹² AAVApo or AAVApoLinkerP144 vg/mice were i.p injected (<i>n</i> = 6/group). Mice were sacrificed at day 15 after tumor cell inoculation and tumor area in the liver was measured quantifying the pixels over a threshold color using Matlab software. Mean±SEM **, <i>P</i><0.01. (D) IFNγ and GM-CSF expression in metastatic nodules in the liver was assessed by RT-PCR. Mean±SEM *, P<0.05. **, P<0.01 (E) Representative pictures of immunohistochemical staining of CD3 in the metastatic nodules in the liver (Magnification: 200×) and quantification of CD3⁺ pixels per field in ten fields per animal quantifying the pixels over a threshold color using Matlab software (Magnification: 100×). Mean±SEM *, P<0.05.</p