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Single cell sequencing of primary patient-derived cells will be carried out for selected

samples using a multiplex specific targeted amplification (STA) reaction and the

Fluidigm array platform, with support of the laboratories of Professor Mel Greaves and

Dr Gareth Morgan at the ICR who have pioneered this approach in haematological


. This will utilise a custom designed assay for each tumour and allow

for the detection of specific mutations and copy number alterations at the single cell

level, upon flow-sorting of patient-derived cultures obtained from biopsy, resection or


By using these complementary approaches and our advanced sampling procedures

we anticipate that we will be able to build up a comprehensive picture of the

evolutionary dynamics of the primary tumour as well as the clonal evolution after the

selective pressures of conventional treatment approaches. Applying evolutionary

biology principles to our analyses, we will be able to assemble phylogenetic trees for

individual tumour growth patterns. This work will be completed within 12 months.

To what extent does genotypic heterogeneity confer phenotypic heterogeneity?

We aim to characterize subclonal phenotypic differences within DIPG tumour samples

and to assess the contributions of genetic heterogeneity to cellular function. We will do

this through the use of advanced image analysis of single-cell derived primary cultures

linked to targeted resequencing of derived colonies as described below.

Advanced high-throughput image analysis of single cell-derived primary cultures

Each prospectively collected tumour specimen will be used to initiate primary cultures

as three-dimensional (3D) neurospheres (NS) and two-dimensional (2D) adherent

stem cell cultures on laminin-coated plates23-25

(serum free, supplemented with growth

factors). Both methods are well established in the Jones lab, with 100% take rate from

DIPG. After initial expansion, patient-derived cells are flow-sorted and a single cell per

well seeded into 96-well laminin-coated plates and round-bottom plates, to allow the

growth of individual 2D and 3D colonies.

Fully automated high-throughput image analysis will be performed in real-time using a

Celigo S. This is a multi-channel brightfield and fluorescence imaging cytometer for

high-throughput cellular image acquisition and processing, extensively validated by

the co-applicant26,27

. Primary single-cell derived clones will be assessed for their

phenotypic heterogeneity in terms of: (a) morphology; (b) clonogenicity/self-renewal

capacity and (c) growth/proliferation. Co-cultures of individual colonies may also be

undertaken to explore interdependence of different subclonal populations. In

preliminary experiments we have observed a variation in self-renewal capacity of

individual samples with clonogenicity ranging from 5-30%, as well as differential

patterns of growth and morphology in single-cell derived clones from the same

primary sample (Figure 2). Cells will be clonally expanded until they reach ~80%

confluence for adherent culture or a size of 700-800µm for NS prior to DNA extraction

for targeted resequencing (below) and subcloning for further comprehensive

downstream functional assays. These will include assessment of stem cell /

differentiation markers and migration/invasion potential in vitro, as well as orthotopic

tumorigenicity in vivo.


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