Dissecting the molecular architecture of the brain tumour stem cell niche
John Lapage, Koentges Lab
CD133L mRNA expression is confined to
regions near vessels
Reclassifying tumour vessels
Abstract
DAPI
CD133L
CD133
vWF
The most common form of primary brain malignancy, Glioblastoma
multiforme (GBM) is a devastating untreatable disease with a median
survival time of only 12 months. Whole GBMs can derive from single
brain tumour stem cells. These also generate the vasculature feeding
the tumour as part of the so-called stem cell niche. The molecular and
cellular architecture of the brain tumour stem cell niche is poorly
known to date. Brain tumour stem cells are positive for CD133
(Prominin 1), a receptor of unknown function. More recently its ligand
has been described in Drosophila and we have cloned its vertebrate
homologue, herein referred to as CD133L, and raised an antibody
against it. This allows us to identify the key components of the stem
cell niche and see where they are localized.
In all stem cell niches known to date, stem cells and their adjoining
cells exchange particular signals that are highly localized to the apical
or basal parts of cells. Recently a novel pathway was discovered which
is responsible for this organization, the Hippo pathway. We are
looking how components of that pathway are deployed inside the
brain tumour stem cell niche.
Novel signalling within the GBM tumour niche
DAPI
CD133
CD133L
Microvascular proliferation is the key hallmark of
late stage GBM. CD133 and CD133L appear to
distinguish different types of structures and vessels
within a GBM in vivo.
Yellow arrow: normal vessel, CD133/CD133L
present and von Willebrand Factor (vWF), an
endothelial marker
Red arrow: nascent vessel, made of strongly CD133+
stem cells only, with patchy CD133L coverage
White arrow: CD133L coats non-stem cells in the
vicinity of stem cells.
DAPI
CD133L mRNA
vWF+
Endothelial Cell
In-situ hybridisation showing CD133L mRNA expression
in a GBM section. CD133L is expressed in endothelial
cells (white arrow) as well as in pericytic cells (yellow
arrow) on the outside of tumour vessels. This suggests
autocrine and paracrine mechanisms to be at play.
Both CD133 and the Hippo pathway define the same stem cells
CD133+ stem cells are surrounded in vivo by other cells with CD133L
signalling ligand on their respective surfaces, facing each other (arrow).
Work supported by the University of
Warwick Chancellor’s Scholarship
and the MLSRF Bursary Fund
DAPI
CD133L
CD133
YAP
Stem Cells have a mixed identity: Endothelial and Pericytic
DAPI
CD133L
CD133
LN-18 GBM cells in 2D culture, showing a single optical section through a
roughly spherical aggregate. CD133 is enriched in morphologically distinct cells
near to the exterior, while CD133L is more tightly confined to the most apical
edges of the structure. The Hippo pathway effector YAP is also enriched in
exterior CD133+ stem cells. YAP is one of the two major transcriptional
coactivators which serve as primary outputs for Hippo pathway activity. Stem
cells and their progeny can self-organise in vitro in patterns similar to their in
vivo niches.
Tight apical co-localisation of
CD133/CD133L in GBM cells
CD133L
Cdc42
CD133
Merge
CD133+
Stem Cell
CD133+ NG2+ vWF+
Mixed identity
tumour stem cells
NG2+ Pericyte
Conclusions
Here we disentangle the brain tumour stem cell niche in vivo. CD133, a
stem cell marker, predicts patient survival in GBMs and other
tumours. We explore its role in vasculogenesis through its previously
unknown ligand, CD133L. We find polarised membrane localisation in
vitro and in vivo. We investigate molecular components of the Hippo
pathway, which is responsible for apical-basal membrane polarity and
a host of signaling complexes localised to such membranes. We
identify its potential relevance in the stem cell niche of GBMs. These
stem cells are of a mixed endothelial and pericytic nature, which might
explain why anti-angiogenic treatments have had no long-term
therapeutic effects.
We are now performing single-cell laser capture/RNA-seq analysis to
localise the particular signaling interactions within the tumour stem
cell niche in vivo.
Acknowledgements
Tight co-localisation of CD133 and
CD133L in apical lamellipodia of
an MO59K GBM cell (Z projection).
This is restricted to the apical
Cdc42+ membrane domain.
Many thanks to Dr Ute Pohl of Queen’s Hospital, the collaborating
pathologist on this project.
Further thanks to Dr Ana Martins, Prof. Georgy Koentges and the rest of
the Koentges Lab (Xintao, Kate, Polly, Sophie and Max).