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(A) Non-specific killing was also excluded by the observation that EGFRvIII-targeting (3C10)-CAR PBMCs did not display significant lysis to LN319 cells. (B) The FACS-sorted CAR-T cells were subjected to RT-PCR. TNF-α was significantly produced in NZ-1 CAR-T cells that were stimulated by LN319 cells, but IL-2 and CD107a were expressed even in mock CAR-T cells, which suggested that the secretion of IL-2 and CD107a was not specific to PDPN antigen.

PDF - 125K, (A) Mice were intraperitoneally injected with different amounts of recombinant adenovirus, and 2 days later, mice were sacrificed by cervical dislocation. The entire peritoneal cavity was fixed with 2% paraformaldehyde/0.2% glutaraldehyde intraperitoneally for 30 min. X-gal staining was performed, and the peritoneal cavity was opened for visual inspection. (B) X-gal-stained organs and mesothelial cells in the peritoneal cavity are shown.

PDf - 67K, Mice were intraperitoneally injected with rAD-Ctrl or rAD-miR-200 and two days later, ES-2 cells were implanted. Three days later, mice were sacrificed and mesenterium was fixed and stained with anti-FN1 antibody and DAPI.

PDF - 11K, Sequence of primers used in the study. Red fonts indicate mutated nucleotides.

The TGFβ-mediated alteration of the tumor microenvironment plays a crucial role in tumor progression. Mesothelial cells are the primary components of the tumor microenvironment for ovarian cancer cells; however, the exact role of TGFβ-stimulated mesothelial cells in ovarian cancer progression remains uncertain. In this report, we examined the effects of TGFβ-treated mesothelial cells on ovarian cancer progression. We show that TGFβ-stimulated human primary mesothelial cells (HPMC) are able to promote cancer cell attachment and proliferation and the activation of the promoter activities of MMP-2 and MMP-9, which are metalloproteinases necessary for tumor invasion. Expression of the miR200 family was downregulated in HPMCs by TGFβ stimulation, and restoration of the expression of miR200 family members in HPMCs suppressed cancer cell attachment and proliferation. Downregulation of the miR200 family by TGFβ induced fibronectin 1 production, which promoted cancer cell attachment to HPMCs. Finally, we demonstrated that the delivery of the miR200s to mesothelial cells in mice inhibited ovarian cancer cell implantation and dissemination. Our results suggest that alteration of the tumor microenvironment by the miR200 family could be a novel therapeutic strategy for ovarian cancer treatment. Mol Cancer Ther; 13(8); 2081–91. ©2014 AACR.

PDF - 151K, Depletion of FN1 does not affect cancer cell proliferation and promoter activity of MMPs by TGFbeta-stimulated HPMCs. (A) HPMCs were transfected with control or FN1 siRNA, and 24 h later, cells were incubated with or without TGFbeta for 48 h. ES-2 and SKOV3 cells that constitutively expressed luciferase were seeded onto the HPMCs . Cells were lysed 24, 48, and 72 h after cell seeding, and luciferase activity was measured. (B) ES-2 and SKOV3 cells were transfected with pGL4, pGL4-MMP-2/promoter or pGL4-MMP-9/promoter together with pRLTK-luc, and 24 h later, the cells were seeded onto HPMCs treated as in (A). The cells were incubated for 24 h, and then, luciferase activity was measured.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in adults with a 5-year overall survival rate of less than 10%. Podoplanin (PDPN) is a type I transmembrane mucin-like glycoprotein, expressed in the lymphatic endothelium. Several solid tumors overexpress PDPN, including the mesenchymal type of GBM, which has been reported to present the worst prognosis among GBM subtypes. Chimeric antigen receptor (CAR)–transduced T cells can recognize predefined tumor surface antigens independent of MHC restriction, which is often downregulated in gliomas. We constructed a lentiviral vector expressing a third-generation CAR comprising a PDPN-specific antibody (NZ-1–based single-chain variable fragment) with CD28, 4-1BB, and CD3ζ intracellular domains. CAR-transduced peripheral blood monocytes were immunologically evaluated by calcein-mediated cytotoxic assay, ELISA, tumor size, and overall survival. The generated CAR T cells were specific and effective against PDPN-positive GBM cells in vitro. Systemic injection of the CAR T cells into an immunodeficient mouse model inhibited the growth of intracranial glioma xenografts in vivo. CAR T-cell therapy that targets PDPN would be a promising adoptive immunotherapy to treat mesenchymal GBM. Cancer Immunol Res; 4(3); 259–68. ©2016 AACR.

Intracellular cytokine staining. Effector cells (2 × 105 cells of mock-transduced PBMCs) were incubated with 4 × 105 target cells (LN319) in 200 μL RPMI-1640 along with GolgiStop in a round-bottom, 96-well plate. Following a 4 h incubation at 37{degree sign}C, the cells were incubated with biotin-SP-AffiniPure F(ab')2 fragment-specific goat anti-mouse IgG at 4{degree sign}C for 30 min. The mock-CAR CD8-T and CD4-T cells did not produce IFN-γ, TNF-α, IL-2, and CD107a.

(A) Tissue array of six organs. PDPN is expressed in the basal layer of the esophagus, in the lymphatic ducts in the stomach, the colon, the rectum and the breast, and alveolar type I cells in the lung. (B) A Public database, BioGPS Dataset Library (http://biogps.org/dataset), show that the expression of PDPN is the highest in placenta with 4-fold value/brain. Error bars, Mean+95%CI. (C) The expression of GBM is 16-fold higher than that of the normal brain according to UCSC Cancer genome browser (https://genome-cancer.ucsc.edu/). Error bars, Mean+95%CI. Normal tissue array (Cybrdi, Inc., Frederick, MD) was deparaffinized and rehydrated. The tissues were then exposed to 3% hydrogen peroxidase for 5 min. An anti-human podoplanin mAb, LpMab-7 (1 ?g/ml) (1) was added to the sections for 1 h at room temperature, followed by EnVision kit (Dako) for 30 min at room temperature. Color was developed using 3, 3-diaminobenzidine tetrahydrochloride (Dako) for 2 min. The sections were counterstained with Mayer's hematoxylin.

Ovarian cancer is a highly invasive and metastatic disease with a poor prognosis if diagnosed at an advanced stage, which is often the case. Recent studies argue that ovarian cancer cells that have undergone epithelial-to-mesenchymal transition (EMT) acquire aggressive malignant properties, but the relevant molecular mechanisms in this setting are not well-understood. Here, we report findings from an siRNA screen that identified the homeobox transcription factor ALX1 as a novel regulator of EMT. RNA interference–mediated attenuation of ALX1 expression restored E-cadherin expression and cell–cell junction formation in ovarian cancer cells, suppressing cell invasion, anchorage-independent growth, and tumor formation. Conversely, enforced expression of ALX1 in ovarian cancer cells or nontumorigenic epithelial cells induced EMT. We found that ALX1 upregulated expression of the key EMT regulator Snail (SNAI1) and that it mediated EMT activation and cell invasion by ALX1. Our results define the ALX1/Snail axis as a novel EMT pathway that mediates cancer invasion. Cancer Res; 73(5); 1581–90. ©2012 AACR.

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PDF file - 46K, Includes information regarding plasmids, In situ hybridization, Immunofluorescence and immunoblot analysis, as well as RT-PCR

XLSX file - 131K, Details of patients' samples are described here