Effect of HDAC and COX-2 co-inhibition on BxPC-3 tumor growth on CAM.

<p>(A) Macroscopic pictures were obtained at the same magnification from bottom and side view. (B) Tumor volume at day 7 after cell implantation. Tumors were treated with 30 µl celecoxib (8 µM), MS-275 (0.2 µM) or drug combination at same concentration. (C) Western-blot detection of COX-2 in 20 µg proteins isolated from tumors grown on CAM and treated with MS-275 (0.2 µM). HSC70 was used as a loading control. (D) Histological aspect of tumors grown on CAM during 7 days and treated with 30 µl celecoxib (8 µM), MS-275 (0.2 µM) or drug combination at same concentration. (E) Western-blot detection of caspase-3 in 40 µg proteins isolated from tumors grown on CAM and treated with MS-275 (0.2 µM) or celecoxib (8 µM). HSC70 was used as a loading control. (F) Ki67 immunostaining and associated quantification of tumors grown on CAM during 7 days and treated with 30 µl celecoxib (8 µM), MS-275 (0.2 µM) or drug combination at same concentration. Results are expressed as mean ± s.d. ***P<.001, **P<.01, *P>.05. n≥3 in each condition.</p

Blood vessel detection in tumors 7 days after BxPC-3 implantation on CAM.

<p>(A) Imaris 3D reconstruction from a 35 µm stacked image after SNA staining (green). Nuclei were counter stained with DAPI (blue). (B) Confocal image after FITC (green) injection in CAM blood vessels. Nuclei were counter stained with TOPRO (blue) (C) Desmin immunodetection (red) in PDAC-CAM stained with SNA (green). Nuclei were counter stained with DAPI (blue).</p

Effect of HDAC and COX-2 coinhibition in PANC-1 and CFPAC-1 cells.

<p>(A) Time-dependent effects of MS-275 and celecoxib on PANC-1 cell growth. (B) Time-dependent effects of MS-275 and celecoxib on CFPAC-1 cell growth. (C) Western-blot detection of Cox-2, p21, p27 in 30 µg CFPAC-1 proteins 48h after 1 µM MS-275 and 10 µM celecoxib treatment. HSC70 was used as a loading control. Results are expressed as mean ± s.d., ***P<.001 versus DMSO or indicated conditions. n≥3 in each condition.</p

Biomarker detection in tumors 7 days after BxPC-3 implantation on CAM.

<p>(A) Western-blot detection of HDAC1, HDAC2, HDAC3, HDAC7, COX-2, TGFBI, MYOF, LTBP2 in 20 µg PDAC-CAM or BxPC-3 proteins. HSC70 was used as a loading control. (B) Immunoperoxydase labelling of MYOF, TGFBI, LTBP2, COX-2.</p

Effect of HDAC and COX-2 coinhibition in BxPC-3 cells.

<p>(A) ELISA assay of PGE₂ in cell culture media 24h and 48h after 1 µM MS-275 and 10 µM celecoxib treatment. (B) Time-dependent effects of MS-275 and celecoxib on cell growth. (C) Time-dependent effects of 1 µM MS-275 and 10 µM celecoxib on apoptotic cell ratio by annexin V/PI flow cytometry and on caspase-3 cleavage. (D) Time-dependent effects of 1 µM MS-275 and 10 µM celecoxib on cell cycle by PI incorporation. (E) Western-blot detection of p21, p27, pRb ppRb and E2F1 in 20 µg BxPC-3 proteins 6 to 48h after 1 µM MS-275 and 10 µM celecoxib treatment. HSC70 was used as a loading control. Results are expressed as mean ± s.d., ***P<.001, **P<.01, *P<.05 versus DMSO or indicated conditions. n≥3 in each condition.</p

Effect of HDAC inhibition on NF-kB activation in BxPC-3 cells.

<p>(A) Effect of an IKK inhibitor (10 µM BAY-11-7082) on 1 µM MS-275-induced COX-2 expression. Phospho-IkBα was used as a control of BAY-11-7082 treatment efficacy. HSC70 was used as a loading control. Densitometry was expressed as a COX-2/HSC70 or IkBα/HSC70 ratio. (B) Western-blot detection of COX-2 in 20 µg BxPC-3 proteins after 1 µM MS-275 treatment and p65 siRNA transfection. HSC70 was used as a loading control. (C) Western-blot detection of p65 in 15 µg BxPC-3 cytoplasm, nucleoplasm or chromatin-associated proteins after 1 µM MS-275 treatment. MEK2 and ORC2 were used as a loading control respectively in cytoplasm and chromatin fractions. Densitometry was expressed as a p65/MEK2 or p65/ORC2 ratio. (D) Time-dependent relative expression of IL-8 mRNA in BxPC-3 cells treated with 1 µM MS-275, 10 µM Celecoxib or a combination of the drugs. Results are expressed as mean ± s.d. ***P<.001, *P<.05 versus DMSO. n≥3 in each condition.</p

Growth curve and immunohistologic characterization of BxPC-3 tumors grown on CAM.

<p>(A) Cells were implanted on CAM at embryonic day 11 and collected 2, 4, 5, 6 or 7 days after implantation. Macroscopic pictures were obtained at the same magnification from top, bottom and side view. Results are expressed as mean ± s.d., n>5 at each time-point. (B) Histologic (Haematoxylin-Eosin or Masson’s trichrome staining) analysis of tumors collected 2, 4, 5, 6 or 7 days after implantation. (C) Immunohistology of tumors 7 days after BxPC-3 implantation on CAM and human PDAC tumors. CK7  =  Cytokeratin-7, CK19  =  cytokeratin-19, CEA  =  Carcinoembryonic antigen, PAS  =  Amylase-periodic acid Schiff staining.</p

Differential proteomic analysis of a human breast tumor and its matched bone metastasis identifies cell membrane and extracellular proteins associated with bone metastasis

The classical fate of metastasizing breast cancer cells is to seed and form secondary colonies in bones. The molecules closely associated with these processes are predominantly present at the cell surface and in the extracellular space, establishing the first contacts with the target tissue. In this study, we had the rare opportunity to analyze a bone metastatic lesion and its corresponding breast primary tumor obtained simultaneously from the same patient. Using mass spectrometry, we undertook a proteomic study on cell surface and extracellular protein-enriched material. We provide a repertoire of significantly modulated proteins, some with yet unknown roles in the bone metastatic process as well as proteins notably involved in cancer cell invasiveness and in bone metabolism. The comparison of these clinical data with those previously obtained using a human osteotropic breast cancer cell line highlighted an overlapping group of proteins. Certain differentially expressed proteins are validated in the present study using immunohistochemistry on a retrospective collection of breast tumors and matched bone metastases. Our exclusive set of selected proteins supports the setup of further investigations on both clinical samples and experimental bone metastasis models that will help to reveal the finely coordinated expression of proteins that favor the development of metastases in the bone microenvironment

Stimulation of osteoclasts and osteoblasts by PC3c cells <i>in vitro</i>.

<p>(<b>A</b>) Primary mouse bone marrow cells were cultured in the presence of RANKL and M-CSF and treated or not (Ct) with conditioned medium obtained from PC3 and PC3c cells. More OCs (white arrow) were formed in cultures treated with PC3c conditioned medium compared to cultures treated with PC3 conditioned medium and Ct (ANOVA, p<0.0001). (<b>B</b>) Primary mouse calvaria cell cultures were treated from day 1-21 with conditioned medium obtained from PC3 and PC3c cell. Mineralized bone nodules were present and visualized by von Kossa staining at day 21 (see mineral in black, white arrows). Mineralized bone nodule formation was decreased when primary cells were treated with conditioned medium from any of the PC3/PC3c cells (compared with non-treated (Ct) cells); the decrease was less when PC3c cell conditioned medium was used (compared with PC3) (ANOVA, p<0.001 versus Ct and versus PC3). (<b>C</b>) PC3 conditioned media stimulated the expression of OPG and RANKL in primary OBs compared with non-treated (Ct) while PC3c conditioned media only inhibits the expression of OPG compared with Ct leading to an higher RANKL/OPG ratio in PC3c conditions. (<b>D</b>) Detection by real-time PCR of SOST, DMP1, OPG and RANKL mRNA expression in MLO-Y4 cells treated with PC3 and PC3c conditioned medium. Results are plotted as the mean number of OC ± SD and OB nodules ± SD of three wells for controls and each condition and are representative of two independent experiments. Genes expression was assessed by real-time PCR on triplicate samples and normalized against that of the ribosomal protein gene L32 *p<0.05; **p<0,001, ***p<0,0001.</p

Accessibilome of Human Glioblastoma: Collagen-VI-alpha‑1 Is a New Target and a Marker of Poor Outcome

Functional targeted therapy has unfortunately failed to improve the outcome of glioblastoma patients. Success stories evidenced by the use of antibody–drug conjugates in other tumor types are encouraging, but targets specific to glioblastoma and accessible through the bloodstream remain scarce. In the current work, we have identified and characterized novel and accessible proteins using an innovative proteomic approach on six human glioblastomas; the corresponding data have been deposited in the PRIDE database identifier PXD001398. Among several clusters of uniquely expressed proteins, we highlight collagen-VI-alpha-1 (COL6A1) as a highly expressed tumor biomarker with low levels in most normal tissues. Immunohistochemical analysis of glioma samples from 61 patients demonstrated that COL6A1 is a significant and consistent feature of high-grade glioma. Deposits of COL6A1 were evidenced in the perivascular regions of the tumor-associated vasculature and in glioma cells found in pseudopalisade structures. Retrospective analysis of public gene-expression data sets from over 300 glioma patients demonstrated a significant correlation of poor patient outcome and high COL6A1 expression. In a proof-of-concept study, we use chicken chorioallantoic membrane in vivo model to show that COL6A1 is a reachable target for IV-injected antibodies. The present data warrant further development of human COL6A1 antibodies for assessing the quantitative biodistribution in the preclinical tumor models