Spheroid cell culture does not induce Oct4GFP or NanogGFP expression in MMTV-PyMT or MMTV-Neu tumor cells.

<p>(A) No GFP+ cells can be detected in spheroids of Oct4GFP+ or NanogGFP+ MMTV-PyMT tumor cells. Oct4GFP+ or NanogGFP+ MMTV-PyMT tumor cells were freshly isolated from tumors, grown as spheres for one passage when pictures were taken. Representative bright field (left side) and fluorescent (right side) pictures are shown. Scale bar = 200 µm. (B-D) Freshly isolated Oct4GFP+ or NanogGFP+ MMTV-PyMT and MMTV-Neu tumor cells were either grown adherently (passage 1–4) or as spheres (passage 0) and analysed for relative expression of (B) GFP, (C) Oct4 and (D) Nanog mRNA by qPCR. mRNA expression of NanogGFP+ testis and compound MMTV-PyMT and MMTV-Neu tumor cells was compared to mRNA expression of control MMTV-PyMT tumor cells, which was set to 1. The analysis was performed in triplicate. The mean ± SEM is shown.</p

Oct4GFP and NanogGFP are expressed in mouse testes.

<p>(A) Cells from testes of GFP−/− control transgenic mice, Oct4GFP+/−, Oct4GFP+/+ and NanogGFP+ animals were isolated and analysed for GFP positivity by FACS. Representative flow cytometry analyses in which sideward scatter is plotted against GFP fluorescence are shown for each genotype. The percentage of GFP+ cells compared to GFP negative control testes is indicated in the gate. (B) GFP mRNA expression in control (n = 3), Oct4GFP+/− (n = 6), Oct4GFP+/+ (n = 2) and NanogGFP+ (n = 5) testes was analysed by qPCR. Circles represent individual samples, the bar indicates the mean value of all samples. (C) GFP+ cells can be detected in Oct4GFP+ and NanogGFP+ testes but not in control testis. Representative pictures of immunofluorescence staining with anti-GFP antibody are shown. Magnification 200×. The framed region is enlarged in the insert.</p

Oct4GFP and NanogGFP expression in inner cell mass of transgenic mouse embryos.

<p>Blastocysts were isolated from pregnant Oct4GFP+ (left side) or NanogGFP+ (right side) mice. Bright field and fluorescence pictures were taken. Arrows indicate GFP+ cells in the inner cell mass (ICM) of the blastocysts.</p

Only very rare GFP+ cells can be detected in compound MMTV-PyMT or MMTV-Neu tumors.

<p>Sections of Oct4GFP+ or NanogGFP+ MT/ret, MMTV-PyMT and MMTV-Neu tumors were stained with an anti-GFP antibody and analysed for GFP positive cells. (A) Representative pictures of GFP immunofluorescence stainings showing the GFP negativity of virtually all sections from the compound tumors. (B) Pictures of the only GFP+ cells detected in single sections from Oct4GFP+ and NanogGFP+ MMTV-PyMT and MMTV-Neu tumors. The framed region is enlarged in the insert. (C) Table summarizing the immunofluorescence analysis, showing the number of analysed tumors per tumor model, the total number of analysed sections, and the total number of detected GFP+ cells per tumor model.</p

GFP mRNA is not expressed in Oct4GFP and NanogGFP transgenic MT/ret, MMTV-PyMT or MMTV-Neu tumors.

<p>Relative GFP mRNA expression levels of (A) MT/ret, (B) MMTV-PyMT and (C) MMTV-Neu compound tumors with indicated genotypes and Oct4GFP+ and NanogGFP+ testes were analysed by qPCR. For each tumor model, GFP mRNA expression levels of compound tumors and Oct4GFP+ and NanogGFP+ testes were compared to one control tumor which was set to 1. Circles represent individual samples, the bar indicates the mean value of all samples. The number of analysed samples is as follows: testes: Oct4GFP+/− n = 7, NanogGFP+ n = 5. (A) MT/ret: control n = 4, Oct4GFP+/− n = 6, Oct4GFP+/+ n = 6, NanogGFP+ n = 6 (B) MMTV-PyMT: control n = 5, Oct4GFP+/− n = 6, Oct4GFP+/+ n = 6, NanogGFP+ n = 6 (C) MMTV-Neu: control n = 5, Oct4GFP+/− n = 5, Oct4GFP+/+ n = 5, NanogGFP+ n = 9.</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

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

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 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