The effects of radiation on the extracellular matrix

Thesis

The extracellular matrix (ECM) is a complex meshwork of insoluble fibrillary proteins and signalling factors interacting together to provide architectural and instructional cues to surrounding cells. Alterations in ECM organisation or composition and excessive ECM deposition have been observed in diseases such as fibrosis, cardiovascular diseases and cancer. Due to its complexity, ECM involvement in cancer is still poorly understood, and its role in ionising radiation response is unknown.

We used subcutaneous mouse models of colorectal and pancreatic cancer to investigate the effects of ionising radiation (IR) on tumour microenvironment. In particular, we looked at the effects of radiation on Col5a1, a regulatory fibril- forming collagen that forms heterotypic fibrils with Col1a1. Immunostainings showed that Col5a1 protein expression decreased in irradiated tumours compared with the non-irradiated controls, whereas Col1a1 expression did not change. This result was confirmed by RT-PCR and Western Blot analysis. Using picrosirius red staining, we found that collagen deposition was significantly altered by irradiation. Besides, five collagen structural parameters (alignment, density, width, length and straightness) were analysed using second harmonic generation imaging, a highly specific technology for detection of collagen fibres. Our data indicated a significant decrease in collagen width after irradiation in a pancreatic cancer model, whereas all parameters were significantly changed in the colorectal cancer model. Col5a1 gene knock-down significantly decreased cell survival upon IR and cell migration as assessed using colony formation assays and 3D-spheroid imaging

To confirm our data, we generated a knockout Col5a1 KPC cell line and injected it subcutaneously in vivo in C57/BL6 mice. The lack of Col5a1 in the in vivo tumour led to a reduction in tumour growth; however, radiation seemed to activate a compensatory effect which refrained the irradiation inhibition effect on tumour growth. In vitro migration assay and cell growth assay, results showed a similar pattern.

Col5a1 knock-down and knockout significantly inhibited cell proliferation, inhibited cell migration in vitro and inhibited tumour growth in vivo. Our data, therefore, present Col5a1 as a possible novel prognostic marker and a potential therapeutic target.

Authors: Diana, A

• DOI: 10.5287/ora-e9zarea48
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English