Date of Award
Master of Science (Human Biology)
School of Medical and Health Sciences
Field of Research Code
Although melanoma is largely curable when detected in its earliest stages, it can metastasise to other tissues, drastically reducing survival rates. The most recent therapies used to treat metastatic melanoma are effective long-term in only 11 to 33% of patients. Our ability to monitor treatment failure is limited. New prognostic markers are urgently required to allow monitoring of treatment response and disease progression.
Circulating tumour cells (CTCs) are released into the bloodstream by the tumours within a patient, this being a key step in melanoma spread. Since CTCs can be detected in the blood of metastatic melanoma patients, these cells can be used as a “liquid biopsy”, providing critical insight into each person’s melanoma biology.
Melanoma CTCs have been described as very heterogeneous, hindering their isolation via commonly used CTC capturing methods. To address this, microfluidic devices have been developed to isolate viable CTCs from blood, independently of their marker expression. This study aimed to determine the effectiveness of two different microfluidic devices (Slanted and A5) in recovering melanoma cell lines, and their potential use in the isolation of CTCs from the blood of metastatic melanoma patients. It also aimed to study additional cancer or melanoma specific markers to be used in immunostaining protocols for detection of CTCs after microfluidic enrichment.
The optimal isolation procedure was identified as two rounds of enrichment with the Slanted spiral device, after which we obtained a 3-log depletion of white blood cells and a recovery of over 60% when cells from two melanoma cell lines were spiked into blood samples from healthy volunteers. In addition, we optimised the detection of CTCs using four melanoma markers (gp100, Melan-A, s100 and MCSP) combined in a multimarker immunocytochemistry staining protocol. The optimised enrichment and detection procedures were validated in a cohort of ten metastatic melanoma patients. Results showed that 40% of the patients had one or more CTCs in their blood (1-4 CTCs/8 mL of blood).
Furthermore, three additional markers (Vimentin, RANK, and ABCB5) were trialled so as to increase detection of highly heterogeneous melanoma CTCs in samples that have been processed through the Slanted microfluidic device.
The improved enrichment and detection of CTCs in the blood of melanoma patients using the methods developed as part of this study will facilitate the molecular, genomic and functional characterisation of melanoma CTCs. This will ultimately improve our understanding of the biology of melanoma CTCs and their role in metastatic spread and treatment response.
Marsavela, A. (2017). Optimisation of the isolation and identification of circulating melanoma cells. https://ro.ecu.edu.au/theses/1993