Figure 2. Decreased expression of MLH1 and PMS2 in multicellular tumour spheroids.

N2a and CHLA-02-ATRT cells were grown for 10 days in 90 mm dishes to generate large numbers of MCTS. MCTS were separated from the bulk monolayer culture using a 19 gauge needle. Spheroids were dissociated using non-enzymatic cell dissociation solution and strained over a 40 µm filter to produce a single-celled suspension. The remaining monolayer cells were scraped from the plate surface and treated as above to produce a single-celled suspension. Half of each of the spheroid and monolayer-derived cells were lysed and total RNA extracted from both cell populations. Reverse transcription PCR was conducted to analyse differences in MLH1 and PMS2 gene expression between the dissociated spheroid (S) and non-spheroid forming monolayer cells (M). A reduced expression of both MLH1 and PMS2 was detected in spheroid cells compared to the bulk monolayer cells. The remaining dissociated spheroid cells and non-spheroid forming monolayer cells were re-seeded as separate populations in new dishes and grown for a further 10 days. RT-PCR was performed on RNA extracted from the entire cell population and analysis revealed a similar decreased expression of MLH1 and PMS2 in spheroid-derived cells compared to monolayer-derived cells. Beta actin expression is shown to confirm equal sample loading.