Uncovering the cellular and molecular mechanisms of radiotherapy soft tissue injury and fat graft treatment

Over half of the 120,000 patients diagnosed with a solid tumour in Australia annually require radiotherapy as part of their treatment. Despite significantly enhancing cancer survival, the damage done to healthy native tissues is inevitable and problematic. Radiotherapy soft tissue injury is progressive, may intensify years after treatment and is characterised by pain, contracture, tissue breakdown, recurrent infection and lymphoedema.

Important implications of radiation injury for surgeons are that surgical procedures (whether for functional restoration or cancer recurrence) in irradiated tissues become technically difficult and more hazardous. Direct wound closure or local flaps are restricted by stiff, non-compliant tissue, and even if wound edges are opposable, they are frequently subject to poor wound healing or breakdown. Further, the ability of an irradiated wound bed to accept skin grafts is diminished, necessitating more complex reconstructive procedures such as free microvascular tissue transfer from distant sites, in which radiation injury is also a chief contributing factor to poor patient outcome.

First, essential cell-specific functions were investigated to establish the effects of radiotherapy-injury on the ability of the cells in skin and subcutaneous tissues to survive or respond to subsequent injury or challenge. Next, molecular alterations resulting from irradiation were characterised at mRNA level using next generation sequencing and further investigated using pathway analysis. Finally, the reparative potential of the adipose-derived stem cells (ADSCs) to reduce radiotherapy injury was investigated in using a similar panel of cellular assays. The constituent proteins secreted by these cells that exerted a regenerative effect were then isolated for further analyses.

The results detailed in this thesis demonstrate that each individual component of skin and subcutaneous tissue exhibits a unique response to radiotherapy injury - challenging the traditional dogma that large scale irreversible cell death is responsible for the manifestation of radiotherapy soft tissue injury. Notable findings include radiotherapy-induced hyper-migration of fibroblasts and pericytes, reduced apoptosis in endothelial and stem cell populations, global suppression of lymphatic endothelial cell (LEC) repair functions and significant alterations in the differentiation capacity of ADSCs.

Next generation gene sequencing revealed key molecular alterations resulting from radiotherapy in a variety of cell types. Significant findings include dysregulation of extracellular matrix proteins and basement membrane collagens – changes likely to contribute to the hypermigratory, adhesive and highly contractile phenotype seen in irradiated fibroblasts. Up-regulation of intercellular adhesion molecule 1 (ICAM-1) in blood vessel endothelial cells found in acute radiotherapy injury was validated in irradiated human tissues and was demonstrated to remain persistently elevated months-to-years after completion of therapy. This finding suggested a key candidate for application to mitigate radiotherapy injury at both the micro and macrovascular level. In order to combat the impaired functions seen in LECs after irradiation, experiments were conducted using stimulation with known potent lymphangiogenic factors VEGF-C and VEGF-D. Irradiated LEC demonstrated an obliterated capacity for response to this stimulation, due to a unique profile of ablated VEGF receptor (VEGFR) -2 signaling and reduced VEGFR-3 activation. Concurrently, up-regulation of interleukin (IL) -8 and chemokine receptor CXCR7 in irradiated LEC was seen and validated in mouse and human tissues to remain upregulated in chronic radiotherapy injury. These two protein candidates, not typically associated with lymphangiogenic properties demonstrated selective lymphangiogenic effect in both normal and irradiated LECs. Together this novel set of data suggest that LECs attempt to regenerate after radiotherapy injury using parallel signaling axes to the traditional VEGF-C and VEGF-D signaling pathways, which are uniquely rendered impotent by radiotherapy injury.

Overall, methods to salvage irradiated tissues to a point to which soft-tissue quality would permit simple wound closure or other tissue repair techniques is desperately needed by clinicians. Fat grafting has been reported as a promising avenue to achieve this when used in previously irradiated areas. It was incidentally noted that irradiated tissue overlying the fat graft became more compliant and less lymphoedematous. The diminished capacity of irradiated ADSC to migrate and differentiate to fat represented significant impairments in their regenerative function. Radiation not only impairs loco-regional ADSC function, but was also shown to block the recruitment and homing of functional ADSC from sites distant to the injury. This may be due to the mentioned presence of CXCR7 secreted by irradiated LEC. Therefore, to overcome injury and aid in regeneration of tissues the mechanical introduction of healthy ADSC, via fat grafting may be needed to override the failed ADSC recruitment mechanisms.

In the fat grafting model using the introduction of the secretome of ADSCs, ADSC-conditioned media (ADSCCM) was able to reverse the effects of radiotherapy injury in both fibroblasts and LEC populations. The final section of this thesis investigated putative therapeutic mechanisms by which ADSCs reverse radiotherapy induced soft tissue injury. Examination of ADSCCM was performed using proteomics, exosome analysis and metabolomics approaches. Several key candidates were identified that may lead to promising therapeutic avenues by which radiotherapy injury can be mitigated.

Understanding of the cellular and molecular mechanisms of radiotherapy induced soft tissue injury, methods by which ADSCCM mediates reversal of the resulting cell dysfunction will provide vital clues and putative therapeutic channels by which to reverse these pathological alterations, thereby reducing the devastating burden of chronic, debilitating side effects of radiotherapy such as fibrosis, lymphoedema and other related diseases, in cancer survivors.