(P 323) AN IN-VITRO INVESTIGATION OF 5-AMINOLEVULINIC ACID MEDIATED PHOTODYNAMIC THERAPY IN SOFT-TISSUE SARCOMA

Objective: Sarcomas are rare, aggressive cancers, occurring most often in the soft tissues of the extremities. The cornerstone of curative margin in soft tissue sarcoma (STS) is typically surgical resection with a wide margin. STS, particularly myxofibrosarcoma (MFS), are often highly invasive, leading to high positive margin and subsequently local recurrence rates. Photodynamic therapy (PDT) describes the administration of a photosensitizer, such as 5-aminolevulinic acid (5-ALA), followed by exposure to light of a particular wavelength. This results in intracellular reactive oxygen species (ROS) production and ultimately cell death. PDT is a recognised treatment in non-melanomatous skin cancer but is not yet established in sarcoma. We believe that it has potential as an intra-operative adjunct following STS resection, prior to wound closure, to kill any remaining cancer cells not resected and reduce the risk of local recurrence. The main aims of this in vitro study were as follows:

1. To quantify the uptake of 5-ALA in STS cells
2. To evaluate the cytotoxic effect of 5-ALA mediated PDT in STS cells
3. To evaluate the mechanism of cell death induced by PDT in STS cells

Methods: MFS (ShefMFS-01 W1) and undifferentiated pleomorphic sarcoma (UPS) (ShefUPS-01) cells were incubated with varying concentrations of 5-ALA (0-4mM) for 4 hours prior to experiments. Uptake of 5-ALA was quantified using flow cytometry to detect cellular fluorescence. PDT was performed by exposing cells to red light (630nm), delivering a total dose of 20J/cm2, using the LEDA-X LED Array (Green Leaf Scientific, Dublin). Cellular viability was measured using a CCK-8 assay at time points of 0, 24, 48 and 72 hours. The production of reactive oxygen species (ROS) was quantified using the CellROX green assay and fluorescence microscopy. Differences between fluorescence, cellular viability and ROS production between treatment groups were assessed using Student’s T-tests, with analysis performed using SPSS.

Results: Both MFS and UPS cells demonstrated uptake of 5-ALA, with significantly higher fluorescence compared to the negative control (cells from the same cell line not incubated with 5-ALA) across all concentrations (p< 0.05). PDT with 5-ALA induced cytotoxicity in both MFS and UPS cells, with significantly decreased cellular viability compared to the control groups (cells not exposed to the light source) at 48 hours (7.5% vs 93.4%, p< 0.0001 and 5.2% vs 79.6%, p< 0.0001 respectively). Cellular ROS were significantly increased in MFS cells undergoing PDT compared to control groups of the same cells not exposed to the light source (p< 0.001).

Conclusion: PDT using 5-ALA induces cell death in STS cells, likely mediated by the production of ROS, demonstrating potential as an intra-operative adjunct in the management of STS. Development of a mouse model will help to ascertain whether its use intra-operatively may result in reduced rates of local recurrence.