FITC
Excitation: 490nm, Emission: 525nm
Background: Cancer-Associated Fibroblasts (CAFs) are significant components of solid malignancies and play central roles in cancer sustainability, invasion and metastasis. In this study we have investigated the invasive capacity and matrix remodelling properties of human lung CAFs after exposure to ablative doses of ionizing radiation (AIR), equivalent to single fractions delivered by stereotactic ablative radiotherapy (SART) for medically inoperable stage-I/II non-small-cell lung cancers.
Methods: CAFs were isolated from lung tumour specimens from 16 donors. Initially, intrinsic radiosensitivity was evaluated by checking viability and extent of DNA-damage response (DDR) at different radiation doses. The migrative and invasive capacities of CAFs were thereafter determined after a sub-lethal single radiation dose of 18 Gy. To ascertain the mechanisms behind the altered invasive capacity of cells, expression of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) were measured in the conditioned media several days post-irradiation, along with expression of cell surface integrins and dynamics of focal contacts by vinculin-staining.
Results: Exposing CAFs to 1 × 18 Gy resulted in a potent induction of multiple nuclear DDR foci (> 9/cell) with little resolution after 120 h, induced premature cellular senescence and inhibition of the proliferative, migrative and invasive capacity. AIR promoted MMP-3 and inhibited MMP-1 appearance to some extent, but did not affect expression of other major MMPs. Furthermore, surface expression of integrins a2, ß1 and a5 was consistently enhanced, and a dramatic augmentation and redistribution of focal contacts was observed.
Conclusions: Our data indicate that ablative doses of radiation exert advantageous inhibitory effects on the proliferative, migratory and invasive capacity of lung CAFs. The reduced motility of irradiated CAFs might be a consequence of stabilized focal contacts via integrins.
This study summarizes the development and testing of a scaffold to promote engraftment of cells in the distal lung. A fibrinogen-fibronectin-vitronectin hydrogel (FFVH) was developed and optimized with respect to its mechanical and biological properties for this application. In vitro, FFVH scaffolds promoted attachment, histiotypic growth and expression of basement membrane proteins by primary ovine lung mesenchymal cells derived from lung biopsies. In vivo testing was then performed to assess the ability of FFVHs to promote cell engraftment in the sheep lung. Treatment with autologous cells delivered using FFVH was clinically well tolerated. Cells labelled with a fluorescent dye (PKH-26) were detected at treatment sites after 1 month. Tissue mass (assessed by CT imaging) and lung perfusion (assessed by nuclear scintigraphy) were increased at emphysema test sites. Post-treatment histology demonstrated cell proliferation and increased elastin expression without scarring or collapse. No treatment-related pathology was observed at healthy control sites. FFVH scaffolds promote cell attachment, spreading and extracellular matrix expression in vitro and apparent engraftment in vivo, with evidence of trophic effects on the surrounding tissue. Scaffolds of this type may contribute to the development of cell-based therapies for patients with end-stage pulmonary diseases.
