Andrea Gervasi

Bioactive Electrospun Fibers of Poly(glycerol sebacate) and Poly(ε‐caprolactone) for Cardiac Patch Application

Scaffolds for cardiac patch application must meet stringent requirements such as biocompatibility, biodegradability, and facilitate vascularization in the engineered tissue. Here, a bioactive, biocompatible, and biodegradable electrospun scaffold of poly(glycerol sebacate)-poly(ε-caprolactone) (PGS-PCL) is proposed as a potential scaffold for cardiac patch application. The fibers are smooth bead free with average diameter = 0.8 ± 0.3 μm, mean pore size = 2.2 ± 1.2 μm, porosity = 62 ± 4%, and permeability higher than that of control biological tissue. For the first time, bioactive PGS-PCL fibers functionalized with vascular endothelial growth factor (VEGF) are developed, the approach used being chemical modification of the PGS-PCL fibers followed by subsequent binding of VEGF via amide bonding. The approach results in uniform immobilization of VEGF on the fibers; the concentrations are 1.0 μg cm(-2) for the PGS-PCL (H) and 0.60 μg cm(-2) for the PGS-PCL (L) samples. The bioactive scaffold supports the attachment and growth of seeded myogenic and vasculogenic cell lines. In fact, rat aortic endothelial cells also display angiogenic features indicating potential for the formation of vascular tree in the scaffold. These results therefore demonstrate the prospects of VEGF-functionalized PGS-PCL fibrous scaffold as promising matrix for cardiac patch application.

Combination of Gefitinib and Pemetrexed Prevents the Acquisition of TKI Resistance in NSCLC Cell Lines Carrying EGFR-Activating Mutation

Introduction: Development of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors is a clinical issue in patients with epidermal growth factor receptor gene (EGFR)‐mutated non–small cell lung cancer (NSCLC). The aim of this study was to investigate the potential of combining gefitinib and pemetrexed in preventing the acquisition of resistance to EGFR tyrosine kinase inhibitors in NSCLC cell lines harboring EGFR exon 19 deletion. Methods: The effect of different combinatorial schedules of gefitinib and pemetrexed on cell proliferation, cell cycle, apoptosis, and acquisition of gefitinib resistance in PC9 and HCC827 NSCLC cell lines and in PC9 xenograft models was investigated. Results: Simultaneous treatment with gefitinib and pemetrexed enhanced cell growth inhibition and cell death and prevented the appearance of gefitinib resistance mediated by T790M mutation or epithelial‐to‐mesenchymal transition (EMT) in PC9 and HCC827 cells, respectively. In PC9 cells and in PC9 xenografts the combination of gefitinib and pemetrexed, with different schedules, prevented gefitinib resistance only when pemetrexed was the first treatment, given alone or together with gefitinib. Conversely, when gefitinib alone was administered first and pemetrexed sequentially alternated, a negative interaction was observed and no prevention of gefitinib resistance was documented. The mechanisms of resistance that developed in vivo included T790M mutation and EMT. The induction of EMT was a feature of tumors treated with gefitinib when given before pemetrexed, whereas T790M was recorded only in tumors treated with gefitinib alone. Conclusions: The combination of gefitinib and pemetrexed is effective in preventing gefitinib resistance; the application of intermittent treatments requires that gefitinib not be administered before pemetrexed.

Lung mesenchymal cells function as an inductive microenvironment for human lung cancer propagating cells

OBJECTIVES The aim of the present study was to characterize the biological properties and in vivo tumourigenic potential of mesenchymal cells (MCs) obtained from non-small-cell lung cancer (NSCLC) samples. METHODS NSCLC samples (53 adenocarcinomas and 24 squamous-cell carcinomas) surgically removed from 46 males and 31 females were processed to identify mesenchymal cells from human lung cancer (hLc-MCs). hLc-MCs were separated from neoplastic epithelial cells, expanded and extensively characterized in vitro. Subsequently, female BALB/c nude mice were subcutaneously injected with either 10(6) or 2.5 × 10(6) Calu-3 (human adenocarcinoma cell line able to reproducibly induce xenografted tumours) alone or in combination with equal doses of hLc-MCs. Control animals were injected with the two doses of hLc-MCs only. RESULTS Primary cultures of hLc-MCs were obtained from >80% of NSCLC specimens. The typical MCs immunophenotype was documented by the expression of CD90, CD105, CD73, CD13 and CD44 at fluorescence-activated cell sorting analysis. CD45, CD14, CD34 and epithelial antigens were negative while CD117 (c-kit) and CD133 (prominin) were partially expressed. Interestingly, nuclear transcription factors octamer-binding transcription factor 3/4 and sex determining region Y-box 2 involved in stemness, thyroid transcription factor 1 in bronchoalveolar commitment, and ETS1 in carcinogenesis, were expressed in hLc-MCs isolated from NSCLC. Specific conditioned media and cocultures confirmed the supportive role of hLc-MCs for cancer cells. In vivo experiments showed that at both doses Calu-3 xenografts doubled in size when hLc-MCs were coinjected. Cell tracking in xenografted tumours, by immunofluorescence combined with fluorescence in situ hybridization analysis, documented hX-chromosome-labelled, Calu-3-derived cytokeratin-positive adenocarcinoma structures surrounded by hLc-MCs. CONCLUSIONS Tumour-propagating cells require the inductive interaction of resident mesenchymal cells to foster lung cancer development.

Isolation and Characterization of Human Lung Lymphatic Endothelial Cells

Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs). Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retain in vitro several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy.