Melanoma cell cluster “nests” formed by cell proliferation

Article: Three-dimensional experiments and individual based simulations show that cell proliferation drives melanoma nest formation in human skin tissue

BMC Syst Biol. 2018; 12: 34.
Published online 2018 Mar 27.
doi: 10.1186/s12918-018-0559-9
https://bmcsystbiol.biomedcentral.com/articles/10.1186/s12918-018-0559-9

Authors:

Parvathi Haridas 1,2, Alexander P. Browning 2, Jacqui A. McGovern 1, D. L. Sean McElwain 1,2
and Matthew J. Simpson 1,2
Affiliations:
1. Institute of Health and Biomedical Innovation, Queensland University of Technology (QUT), Kelvin Grove, 4059, Australia
2. School of Mathematical Sciences, QUT, Brisbane, 4001, AustraliaFunders:

Funding

This study was supported by research funds from the Australian Research Council (DP170100474)

Aim:

Melanoma nests are a group of cells on the skin used as a diagnostic marker for skin cancer. More severe skin cancers usually present with larger nests.
It has been suggested that melanoma nests are created by one of two cellular processes: cell populations growing by division (mitosis) or by many cells meeting together (migration).
There has been some debate over which process is more responsible for building these nests.
The research team hoped to better understand the core biology of melanoma nests using a range of 2D/3D experiments on human skin and individual based simulations.

Terminology:

MTT assay: The MTT assay is a colorimetric assay for assessing cell metabolic activity
Melanoma: Form of skin cancer where the pigment producing cells (melanocytes) become mutated and grow quic3D human skin modelkly
Melanoma nest: Group of cancerous melanocytes
Cell migration: Important process that allows cells to move around and perform various functions including wound repair and immune responses
Cell proliferation: Process where a cell grows and splits to form two daughter cells which results in the increase of cell numbers
3D human skin model: Construction of human skin with a range of human skin cell types
Individual based model: Simulation of events within a group
Irradiation (to cells): Stops cells being able to produce more cells by way of mitosis

Method:

Human skin samples were obtained ethically from patients undergoing elective plastic surgery. Different cell types in the skin (keratinocytes, fibroblasts) were isolated and cultured from this tissue as well as cells from the human melanoma cell line SK-MEL-28. A portion of these cells were gamma-irradiated to stop cell proliferation.
Irradiated and non-irradiated melanoma cell groups were then cultured and examined with barrier assays for cell proliferation and migration. Proliferation was detected by tallying cell numbers during a 24-hour period. Migration was inferred by increased diameter of spreading cell populations.

3D human skin experimental model
Components of the donated skin tissue were used to create a three-dimensional skin model. The top layer of skin was removed (epidermis) and sterilised 3mm rings placed in the dermis papillary layer. Primary keratinocytes, primary fibroblast cells and either non-irradiated or irradiated melanoma cells were implanted into this layer in medium.

This structure was incubated for two days followed by ring removal, medium replenishing and then two further days of incubation. After four days total, a range of tests were performed to understand how cells were distributed.

An MTT assay identified cells with high metabolic activity (cancer cells). A greater concentration of metabolically active cells related to darker purple colouration/nests.
Melanoma cells were further identified with antibodies (immunohistochemistry).

Individual based simulation (IBM)
A cyber IBM was then constructed to check the results of the 3D skin tissue experiment. This involved making a computerised 3D lattice 3mm x 3 mm and depth 2 mm to reflect the central section of the experimental skin model. Skin and melanoma cells designed to imitate real cells were placed into the surface of the lattice. Cells, referred to as “agents”, were placed in their own site in the lattice with 20 um between each agent. The number of cells roughly matched the those in the 3D human tissue model.
A mathematical algorithm was developed for this simulation to investigate the probability events of cells migrating or proliferating.

Results:

When melanoma cells were irradiated, this stopped cells being able to reproduce but they could still move around. In comparison, non-irradiated melanoma showed skill in both migration as well as proliferation. The latter (proliferation) seemed to push nest formation which was much greater in the models using non-irradiated melanoma cells.
Larger nests were supported by having more cells to begin with and cell proliferation.
IBM simulation also showed that melanoma nests were formed mostly through cell proliferation.

Conclusion:

Cell proliferation, rather than migration, is the main stimulator of melanoma nests. Likewise, nest size is driven by proliferation but also through having a greater number of melanoma cells to start with.
These findings from 3D skin experiments are reinforced by the IBM simulation.

Relevance:

Skin cancer is one of the most common cancers in Australia, with two in three Australians reported to be diagnosed with the cancer by the time they are 70 (1).
Melanoma is the deadliest type of skin cancer and so finding new therapies and drugs is a big focus of current research.
New treatments are usually developed from understanding the essential biological principles that cause the disease. This study narrows down the cellular process most responsible for melanoma nest formation – proliferation. Stopping nest creation by exploiting proliferation pathways could be a novel therapeutic target. Thus, having potential to stop the disease progressing or mortality.

HRA Comment:

Animals skin models are frequently used to study skin diseases and the side effects from pharmaceuticals or cosmetics designed for human use. A significant barrier in this approach are the biological differences in skin structure between humans and animals. For example, in humans, certain layers of the skin and hair growth patterns are different compared to other animals. These qualities could impact the relevancy of experimental setup and results of animal skin compared to humans.
As well as this, there are some challenges that have arisen from human cancer research in animal models. Implanting human tumours in mice is not always successful and has resulted in a large and wasteful number of animals being tested on. Similarly, if the cancer tissue were lucky to implant successfully, then there have been further reports of human cells being replaced with mouse cells. This means that cancer tissue is no longer entirely human, and this could affect results and their ability to translate meaningfully to humans (2)

References:

1 https://www.cancer.org.au/cancer-information/types-of-cancer/skin-cancer
2 https://dmm.biologists.org/content/10/8/939

Although this study does not directly use any animals it was noted that serum and antibodies with animal hosts were utilised

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