How benign mole melanocytes can switch from a stable state to a cancerous melanoma

Did you know that in Australia, one person dies from melanoma every five hours?

Melanoma is often referred to as ‘Australia’s National Cancer’ and is the third most common cancer in Australia¹. Most are caused by prolonged and repeated exposure to UV radiation in sunlight, but there are some melanomas that are not sun related, eg, Bob Marley famously died of an acral lentiginous melanoma on his toe in 1981². Melanocyte cells produce melanin to protect the deeper layers of skin but when unregulated or uncontrolled, melanomas can develop, metastasise and become life threatening.

A team of researchers working at University of California, San Francisco, in collaboration with The Huntsman Institute, Utah aimed to investigate the mechanisms by which a benign mole (nevus) can switch from being stable melanocyte cells to cancerous melanoma cells³.

Groundbreaking results come to light

BRAF^V600E is a genetic mutation present in 80% of benign mole (nevus) melanocytes which has been shown to be the driver of 50% of all cutaneous melanomas³. The gene doesn’t automatically cause the development of a tumour but is believed to drive both rapid proliferation and the subsequent arrest stage that forms a nevus. Initial studies focused on characterising the growth and proliferation profiles of BRAF^V600E induced melanocyte cells. Subsequent investigations looked at primary nevi cells which had been transduced with an mCherry linked AURKB mutation (a downstream gene of BRAF^V600E). Differential proliferation profiles were studied between cells carrying the mutation, and those without it.

At the outset, many commonly used imaging techniques presented several challenges for this study particularly given the limited sample size. Devices that only measure population-level metrics were limited to a single data point per well, making it difficult to obtain reliable, robust results with the limited available cells. Commonly used fluorescence imaging and labelling introduce phototoxicity, which disrupts the growth and proliferation of cells and can cause cell death. The requirement to use fluorescent imaging to confirm transduction could consequently induce abnormalities in the growth and proliferation profiles of the cells.

Livecyte: Perfectly suited to resolving these issues and more

Livecyte⁴ presents high-contrast, label-free images where pixel intensity is directly proportional to cell dry mass — a unique property that measures the amount of matter in each cell. This enables robust cell segmentation and tracking leading to accurate single-cell measurements of proliferation, amongst others. Dry mass quantification facilitates the analysis of cellular growth profiles, not possible using other imaging techniques.

Livecyte treats every individual cell as a separate data point. Therefore, reliable and significant cell proliferation data could be extracted from the small number of primary nevi cells. This would not be possible if treating each well as a single data point, as high-throughput analysers do.

Livecyte demonstrates the importance of measuring growth and proliferation independently

Using Livecyte the research group discovered that induction of BRAF^V600E using increasing doses of doxycylin lead to a reduced cell count, and quite unexpectedly, an increase in cell dry mass. The results indicate that growth arrest induced by BRAF^V600E is not by oncogene induced senescence (OIS), as expected in the literature, but instead by reversible mitotic cell cycle arrest.

Figure 1: Cell proliferation and growth rates upon dosing with BRAF^V600E inducing doxycycline.

Livecyte revealed that the mCherry-expressing cells proliferated at a far greater rate than non-mCherry-expressing cells. This indicates that AURKB is an integral player in the conversion of stable nevi melanocytes into melanoma.

Figure 2: mCherry-expressing cells with AURKB mutation were released from miRNA regulation and proliferated faster than those without the AURKB mutation.

Livecyte is a powerful label-free cell imaging and analysis tool that automatically measures cell count, dry mass and mitosis in 96-well plate assays. This enables cell proliferation to be quantified independently from cell growth factors, giving a more in-depth analysis of cell proliferative behaviours. Livecyte presents clear advantages to a researcher in the accuracy of measurements, as well as saving a considerable amount of time and energy.

References

1. https://www.melanomaresearch.com.au/
2. https://www.abc.net.au/everyday/how-to-check-for-skin-cancer-dark-skin-people-of-colour/100709432
3. https://www.phasefocus.com/resources/customer-testimonials/huntsman-cancer-institute-plasticity-melanocytes-during-development-melanoma
4. https://www.atascientific.com.au/products/phasefocus-livecyte/

Top image credit: ©stock.adobe.com/au/Damian Gretka