Oral Presentation 2015 Annual Meeting of the Australasian Society for Dermatology Research

Isolation of cells from different subcompartments of melanoma 3D spheroids (#19)

Farzana Ahmed 1 , Kimberley A Beaumont 2 , Crystal A Tonnessen 1 , Wolfgang Weninger 2 3 , Nikolas K Haass 1 2 3
  1. The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane
  2. Centenary Institute, Newtown, NSW, Australia
  3. Discipline of Dermatology, University of Sydney, Camperdown, NSW, Australia

Despite the recent successes in melanoma therapy there is still no cure for metastatic melanoma, mainly due to the acquisition of cancer drug resistance. Therefore it is of priority to investigate potential mechanisms of resistance. It is thought that drug resistance is conferred by dormant, slow growing cells that reside in the inner core of the heterogeneous tumour. Cells in this hypoxic and nutrient-deprived microenvironment remain G1 arrested and thus may evade drug action. On the other hand cells in the periphery of a melanoma are more likely to be killed by the drug action, leading to the effect of transient remission.

This project aims to elucidate the molecular differences between the G1-arrested inner and proliferating outer tumour cell populations. We used our in vitro 3D spheroid model that has been shown to mimic the situation of a tumour in vivo more faithfully than 2D cell culture. The fluorescent ubiquitination-based cell cycle indicator (FUCCI) allowed us to distinguish between the G1-arrested inner from the proliferating outer zone of the spheroid. We used Hoechst dye diffusion to mark the outer population. To do that we optimised the Hoechst dose and incubation time so that by confocal microscopy the Hoechst-positive zone correlated with the proliferating zone. This allowed us to separate outer from inner cells by FACS based on Hoechst-positivity. We have already shown the differential protein expression pattern of markers such as MITF, SLUG, Vimentin, E- and N-Cadherin in inner and outer spheroid populations. We use these markers as quality control.

Now we are at phase of analyzing the transcriptome and proteome of the inner and outer populations. The ultimate goal of the project is to understand the detailed biology of tumour heterogeneity in melanoma and to utilize the knowledge to design better therapeutics against this deadly disease.