Author Identifier
Sanjeev Adhikari: http://orcid.org/0000-0003-4704-9744
Date of Award
2025
Document Type
Thesis
Publisher
Edith Cowan University
Degree Name
Doctor of Philosophy
School
School of Medical and Health Sciences
First Supervisor
Elin Gray
Second Supervisor
Aaron Beasley
Third Supervisor
Fiona Pixley
Abstract
Circulating tumour DNA (ctDNA) has received enormous attention over the last decade owing to its biomarker potential in cancer detection, monitoring, and genotyping. Despite substantial advancements made in these fronts, little is known of the mechanisms/factors that influence ctDNA released by tumours. The lack of mechanistic framework to relate the biological factors associated with ctDNA release has posed a challenge to interpreting ctDNA results in clinical settings. Here, we ctDNA released from six pairs of BRAF inhibitor- sensitive and matching resistant melanoma cell lines in vitro, and two pairs in vivo, examining factors like apoptosis, necrosis, and proliferation that could potentially affect ctDNA levels. Additionally, we assessed the impact of baseline and cytokine-activated mouse and human-derived macrophages on ctDNA levels produced by melanoma cells in vitro, developing self-optimised co-culture experiments. Finally, xenograft tumour models were evaluated for melanoma cell-intrinsic factors and infiltrating tumour macrophages as modulators of plasma ctDNA.
In in vitro models, five out of the six cell pairs (83.3%) demonstrated a statistically significant increase in ctDNA shedding in BRAF inhibitor-resistant melanoma cells compared to their sensitive counterparts. The main difference in ctDNA levels between these cell pairs was in the amount of high molecular weight ctDNA released, followed by mononucleosomal and polynucleosomal ctDNA. The elevation in ctDNA was consistently associated with the elevation of apoptosis and necrosis, varying in a cell-specific manner. Notably, there was a strong positive correlation between changes in ctDNA over time and cell proliferation.
Protocols for in vitro co-culture experiments of mouse and human-derived baseline and cytokine-activated macrophages with melanomas were developed to investigate the role of macrophages on ctDNA levels. Results consistently showed reduced ctDNA levels in cultures with macrophages, which was associated with their phagocytic capacity.
Xenograft models showed differences in ctDNA release between the two sensitive-resistant melanoma pairs tested. BRAF-inhibitor resistant M249 R4 melanoma cells demonstrated significantly lower release of ctDNA compared to sensitive M249, consistent with their release pattern in vitro. The other pair, SKMel28 and its resistant counterpart SKMel28 BR9, showed no difference in ctDNA shedding. Notably, the resistant cell SKMel28 BR9 xenografts exhibited significantly higher number of macrophages than SKMel28 tumours, suggesting these macrophages might consume ctDNA, resulting in lower levels in circulation, in contrast to in vitro settings- where the TME was absent. Indeed, a strong negative correlation between the macrophage infiltrate and ctDNA levels was observed for SKMel28 BR9 tumours, further supporting the role of macrophages in modulating ctDNA shedding into the circulation.
These findings underscore the notion that cellular and TME features may influence ctDNA release into circulation, and therefore impacting diagnostic tests based on ctDNA analysis. In particular, macrophages were found as a key biological factor that affect ctDNA levels in circulation. Overall, studying ctDNA biology is essential to solidify its role as a powerful liquid biopsy tool.
DOI
10.25958/cdqr-dn53
Access Note
Access to this thesis is embargoed until 12th August 2026
Recommended Citation
Adhikari, S. (2025). Investigating the biological determinants influencing the release of circulating tumour DNA. Edith Cowan University. https://doi.org/10.25958/cdqr-dn53