PDGFR-α expression in invasive breast carcinomas by immunohistochemistry (streptavidin-biotin-peroxidase)

<p><b>Copyright information:</b></p><p>Taken from "Overexpression of platelet-derived growth factor receptor α in breast cancer is associated with tumour progression"</p><p>Breast Cancer Research 2005;7(5):R788-R795.</p><p>Published online 1 Aug 2005</p><p>PMCID:PMC1242156.</p><p>Copyright © 2005 Carvalho et al.; licensee BioMed Central Ltd.</p> Platelet-derived growth factor receptor α (PDGFR-α) expression in pericytes and smooth muscle cells of a blood vessel: internal control (original magnification × 200); Absence of PDGFR-α expression in neoplastic cells (original magnification × 200); PDGFR-α diffuse cytoplasmic expression in neoplastic cells (original magnification × 200; inset × 400); Neoplastic cells showing strong and diffuse cytoplasmic PDGFR-α expression (original magnification × 400)

Biofunctional Nanofibrous Substrate Comprising Immobilized Antibodies and Selective Binding of Autologous Growth Factors

The immobilization of biomolecules at the surface of different biomedical devices has attracted enormous interest in order to enhance their biological functionality at the cellular level. This work aims to develop a biofunctional polymeric substrate capable of selectively binding growth factors (GFs) of interest from a pool of proteins present in a biological fluid: platelet lysate (PL). To achieve this goal, the surface of electrospun PCL nanofibers needs to be activated and functionalized to be able to insert chemical groups for the immobilization of antibodies. After determining the maximum immobilization capacity of each antibody, TGF-β1 (12 μg mL^–1), bFGF (8 μg mL^–1), and VEGF (4 μg mL^–1), the next step was to confirm their bioavailability using recombinant proteins. The binding efficiency of PL-derived GFs was of 84–87% for TGF-β1, 55–64% for bFGF, and 50–59% for VEGF. Cellular assays confirmed the biological activity of the bound VEGF (both recombinant and PL-derived). Multiple antibodies (i.e., bFGF and VEGF) were also immobilized over the same structure in a mixed or side-by-side fashion. Using both autologous biological fluids and cells, it is possible to use this platform to implement very effective and personalized therapies that can be tailored to specific medical conditions

Flow cytometry analysis of hASCs.

<p>The expression pattern of specific antigens on the surface of the hASCs is depicted with representative histograms and the expression of each marker. The cell population expressed CD29, CD44, CD73 and HLA-ABC, and did not express CD34, CD45 and HLA-DR.</p

RT-PCR analysis of VEGFR2 mRNA expression during endothelial differentiation on the electrospun PHB/PHB-HV fiber mesh and TCPS.

<p>Total RNA was extracted from cells cultured on basal medium and endothelial differentiation medium for analysis of VEGFR2 mRNA expression. The cells were cultivated up to 21 days.</p

Protein expression of hASCs during endothelial differentiation.

<p>Confocal images of the expression of the VE-Cadherin (A) and the vWF factor (B) after 21 days. A.1 and B.1 - hASCs cultured with the basal medium on the TCPS coverslips, A.2 and B.2 - hASCs cultured with the endothelial differentiation medium on the TCPS coverslips, A.3 and B.3 - hASCs cultured with the basal medium on the electrospun PHB/PHB-HV fiber mesh, A.4 and B.4 - hASCs cultured with the endothelial differentiation medium on the electrospun PHB/PHB-HV fiber mesh. Scale bar 20 µm. The images, A.2, A.4, B.2 and B.4 represent the overlay of bright-field and confocal images for visualization of the fiber mesh. (C) Fluorescence intensity of the expression of VE-Cadherin and the vWF factor in cells differentiated on TCPS coverslip and electrospun PHB/PHB-HV fiber mesh. The results are expressed as the means ± SD, (*) significant difference for p<0,05. (a.u): arbitrary units.</p

Proliferation and viability of hASCs cultured on TCPS and electrospun PHB/PHB-HV fiber mesh.

<p>(A) MTT proliferation assays performed 7, 14 and 21 days after the cell seeding and cultured in two specific medium: the basal medium and the endothelial differentiation medium. The results are expressed as the means ± SD, (*) indicating a significant difference with p<0.05, (**) p<0,01, (***) p<0,001; (B) cell viability after 21 days of cell culture with the basal medium and (C) the endothelial differentiation medium on the electrospun PHB/PHB-HV fiber mesh, as analyzed by Calcein-AM staining.</p