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Nuclear FAK Controls Chemokine Transcription, Tregs, and Evasion
of Anti-tumor Immunity
Alan Serrels,1,2 Tom Lund,1 Bryan Serrels,1 Adam Byron,1 Rhoanne C. McPherson,2 Alexander von Kriegsheim,1 Laura Gómez-Cuadrado,1 Marta Canel,1 Morwenna Muir,1 Jennifer
E. Ring,3 Eleni Maniati,4 Andrew H Sims,1 Jonathan A. Pachter,3 Valerie G. Brunton,1 Nick Gilbert,5 Stephen M. Anderton,2 Robert J.B. Nibbs,6 and Margaret C. Frame1
1Edinburgh
Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom. 2MRC Centre for Inflammation Research, The Queens Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom.3Verastem Inc, 215
First Street, Suite 440, Cambridge, Massachusetts, United States of America. 4Queen Mary, University of London, Centre for Cancer and Inflammation, Charterhouse Square, London, United Kingdom. 5MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh,
Edinburgh, United Kingdom. 6Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow, United Kingdom.
D!
400
Volume (mm3)
SCC FAK-wt
SCC FAK-/SCC FAK-/- CD4 Depletion
SCC FAK-/- CD8 Depletion
SCC FAK-/- CD4 + CD8 Depletion
SCC FAK-/- Isotype Ctrl
300
Volume (mm3)
400
****
****
200
****
100
SCC FAK-wt
SCC FAK-/SCC FAK-wt CD4 Depleted
SCC FAK-wt CD8 Depleted
SCC FAK-wt CD4 + CD8 Depleted
SCC FAK-wt Isotype Ctrl
300
****
****
200
****
100
****
0
3
7
10
14
Days
E!
Volume (mm3)!
150
100
50
0
150
100
50
0
400
Pre-challenge:!
SCC FAK-/-!
Tumor!
Free!
Pre-challenge:!
SCC FAK-/-!
17
21
0
Tumor!
Free!
3
7
F!
400
Re-challenge:!
SCC FAK-/-!
Volume (mm3)
0
0
Re-challenge:!
SCC FAK-wt!
SCC FAK-wt Ctrl
10
14
Days
SCC FAK-wt
SCC FAK-/SCC FAK-kd
17
21
****
300
***
200
100
SCC FAK-/- Ctrl
200
0
0
7
14
21 28
Days!
35
42
0
49
0
3
7
10 14 17 21 24 28
Days
Figure 1. Key points:
•  FAK expression is required for the survival, and growth, of SCC tumors in FVB host mice with a functional
adaptive immune system.
•  CD8+ T-cells are responsible for regression of FAK-/- tumors
•  FAK-wt SCC tumors have similar immunogenicity to FAK-/- SCC tumors, and that their distinct fates are not
due to differences in intrinsic ability to prime an adaptive immune response.
•  Tumor survival depends on FAK kinase activity.
20
10
10
5
60
50
*
SCC FAK-/-!
*
40
30
10
0
SCC FAK-kd!
SC K
C -w
S C FA t
K
C
F A -/K
-k
d
FA
CD44hiCD62Llow!
CD44hiCD62Lhi!
CD44lowCD62Llow!
%CD8+
%Ki-67+
L!
SCC FAK-wt!
40
C
C
SC FAK
S C C F -wt
C AK
F A -/K
-k
d
K!
0
60
0
SC
%CD8+
PD-1+Tim-3+LAG-3+
80
20
K
-w
FA t
C
K
F A -/K
-k
d
30
C
SC
0
40
F
SC AK
C -w
SC F t
C AK
F A -/K
-k
d
SC K
C -w
SC F t
C AK
F A -/K
-k
d
0
15
0
FA
C
SC
C
SC
SC
C
K
-w
FA t
K
F A -/K
-k
d
0
20
C
0
5
1
CD45+ CD3+
CD4- CD8+
100
SC
1
2
% CD8+
*
%CD8+CD44hi
% CD8+CD44hi
50
**
10
2
J!
120
40
FA
15
3
SC
PD-1+Tim-3+
80
60
C
F
SC AK
C -w
SC F t
C AK
F A -/K
-k
d
SC K
C -w
S C FA t
K
C
F A -/K
-k
d
FA
C
I!
F!
*
C
PD-1+LAG-3+
SC
C
SC FAK
S C C F -wt
C AK
F A -/K
-k
d
H!
*
CD69+
E!
SC
0
SC
FA t
K
F A -/K
-k
d
C
SC
SC
C
-w
K
FA
C
SC
Fold Change
(normalized to CD8+ T-cell No.)
effCD8+ T-cell
3
FA
20
0
0
C
40
20
5
SC
60
%CD45+
40
4
SC
10
80
%CD4+
%CD4+
%CD45+
15
G!
100
60
CD45 CD3
CD4-CD8+
D!
120
+
SC
80
20
CD45+ CD3+
CD4+ CD8-
C!
+
C
F
SC AK
C -w
SC F t
C AK
F A -/K
-k
d
25
CD69+
B!
CD44hiCD62Llow!
CD44hiCD62Lhi!
CD44lowCD62Llow!
CD45 CD3
CD4+CD8-
A!
%CD8+CD44hi
+
SC
+
Figure 2. FAK-depleted tumors exhibit a heightened CD8+ Tcell response. (A) FACS quantification of total intra-tumoral CD4+
T-cells. (B) FACS quantification of CD69+ cells as a percentage of
CD4+ T-cells. (C) FACS quantification of CD4+CD44hiCD62Llow,
CD4+CD44hiCD62Lhi, CD4+CD44lowCD62Lhi T-cell subpopulations.
(D) FACS quantification of total intra-tumoral CD8+ T-cells. (E)
FACS quantification of CD69+ cells as a percentage of CD8+ Tc e l l s . ( F ) Q u a n t i fi c a t i o n o f C D 8 + C D 4 4 h i C D 6 2 L l o w ,
CD8+CD44hiCD62Lhi, CD8+CD44lowCD62Lhi T-cell subpopulations.
(G) Fold-change in effector (CD8+CD44hiCD62Llow) CD8+ T-cells
when normalized to total CD8+ T-cell percentage. (H) FACS
quantification of PD-1+LAG-3+ T-cells as a percentage of
CD8+CD44hi tumor infiltrating T-cells from 6 independent tumor
samples 7 days post implantation.
(I) FACS quantification of
PD-1+Tim-3+ T-cells as a percentage of CD8+CD44hi tumor
infiltrating T-cells from 3 independent tumor samples 7 days post
implantation. (J) FACS quantification of PD-1+Tim-3+LAG-3+ T-cells
as a percentage of CD8+CD44hi tumor infiltrating T-cells from 3
independent tumor samples 7 days post implantation. (K) FACS
quantification of Ki-67+ cells as a percentage of tumor infiltrating
CD8+ T-cells from 3 independent tumor samples 7 days post
implantation. (L) Representative images of histological staining of
frozen sections from SCC FAK-wt, SCC FAK-/-, and SCC FAK-kd
tumors 7 days post implantation using anti-CD8 antibody clone
53-6.7. Dashed white lines demark tumor boundary. Scale bar =
500 μm. Data are represented as mean ± SEM. N = 5 unless stated
otherwise. p-value = Not significant >0.05, * <0.05, ** <0.01, ***
<0.001, **** <0.0001.#
!
Figure 2. Key points:
•  SCC FAK-/- and FAK-kd (kinase-deficient) tumors have elevated effector CD8 T-cell numbers.
•  CD8 T-cells infiltrating SCC FAK-wt tumors have elevated expression of markers indicative of a heightened
state of dysfunction.
E!
K
Control!
C
FA
C
F!
SCC FAK-wt
SCC FAK-/SCC FAK-wt CD25 Depletion
SCC FAK-wt Isotype Ctrl
250
CD4+CD25+FoxP3+!
Thymus!
SCC FAK-wt!
200
150
100
Helios!
50
0
0
3
7
10
Day
14
17
21
SC
F! 500
0.5
Cytoplasm!
0
300
Figure 4. Key points:
•  FAK drives expression of chemokines and cytokines in SCC cancer cells.
•  Network analysis of the relationship between FAK-dependent chemokine ligand expression in SCC cells
and tumor-infiltrating Treg chemokine receptor expression shows the existence of a FAK-driven paracrine
signaling axis between cancer cells and intra-tumoral Tregs based on chemokine ligand-receptor
interactions.
•  Ccl5, Cxcl10, and TGFb2 expression are dependent on FAK kinase activity.
Total!
FAK!
GAPDH!
PARP!
FAK!
200
100
GAPDH!
PARP!
Total!
0
0
3
7
10 14 17 21 24 28
Days
t!
e!
yt
-w
c
K
o
FA
tin
C
ra
e
C
K
S
! ! ! !
C Nuc C Nuc
W
W
FAK (60 Sec)!
FAK (10 min exp.)!
PARP!
GAPDH!
FAK!
Tubulin!
Figure 5. Nuclear FAK regulates transcription of Ccl5 which is
required for Treg recruitment and tumor growth. (A) qRT-PCR
analysis of Ccl5 gene expression knockdown in SCC FAK-wt cells
stably expressing two independent shRNA constructs targeting
Ccl5 (P1 and P2). (B) SCC FAK-wt shRNA-Ccl5 tumor growth in
FVB mice. N = 6 tumors / group. (C) FACS quantitation of tumor
infiltrating Treg absolute numbers from SCC FAK-wt shRNA-Ccl5
tumors.
Data represents a single value from 6 pulled tumor
samples. (D) Anti-FAK western blot of cytoplasmic, nuclear, and
total protein fractions from SCC FAK-wt, SCC FAK-/-, and SCC
FAK-NLS cells.
Anti-GAPDH (cytoplasmic) and anti-PARP
(nuclear) antibodies were used as controls to check the quality of
cellular fractionation. (E) qRT-PCR analysis of Ccl5 gene
expression in SCC FAK-NLS mutant cells. (F) Tumor growth of
SCC FAK-NLS expressing cells in FVB mice. (G) Anti-FAK western
blot from whole cell (WC) and nuclear (Nuc) lysates extracted from
SCC FAK-wt tumor cells and primary skin keratinocytes. 60 second
exposure time is shown for all samples and additional 10 minute
exposure time is shown for keratinocyte samples. Anti-GAPDH
(cytoplasmic) and anti-PARP (nuclear) antibodies were used as
controls to check the quality of cellular fractionation. Data are
represented as mean ± SEM except when detailed otherwise. pvalue = Not significant >0.05, * <0.05, ** <0.01, *** <0.001, ****
<0.0001. !
!
Figure 5. Key points:
•  Depletion of Ccl5 expression in FAK-expressing SCC cells results in tumor regression associated with
decreased intra-tumoral Treg levels.
•  FAK-dependent expression of Ccl5 requires its nuclear localization and kinase activity.
•  FAK nuclear accumulation is specific to SCC cancer cells.
Figure 3. Key points:
•  Regulatory T-cell (Treg) numbers are increased in FAK-expressing tumors.
•  Loss of FAK or FAK kinase activity alters the CD8 T-cell : Treg ratio in tumors, shifting the balance from
tolerance to immunity.
•  Tregs are required for survival of FAK-expressing SCC tumors.
•  Tregs are likely recruited into FAK-expressing tumors. Helios positivity implies Tregs are of thymic origin.
Figure 4. FAK regulates transcription of cytokines implicated
in Treg recruitment and expansion. (A) Transcriptomic profiling
of SCC FAK-wt and SCC FAK-/- cells. (B) Functional enrichment
analysis of genes upregulated in SCC FAK-wt cells (bottom gray
bar in A). Overrepresented biological processes are displayed as a
heat map (log10-transformed color scale) (top); asterisks indicate
presence of cytokine-related genes. Overrepresented gene families
are displayed as a bar chart (bottom); dashed line indicates P =
0.001. Displayed terms satisfy P < 0.05 (Benjamini–Hochbergcorrected hypergeometric tests). (C) qRT-PCR array analysis of
cytokine and chemokine expression in SCC FAK-wt and SCC
FAK-/- cells. Gray bar indicates cluster of genes upregulated in
SCC FAK-wt cells; cytokine and chemokine gene names are listed.
Green arrowheads indicate reported roles in Treg recruitment; red
arrowhead indicates reported role in peripheral Treg induction. (D)
qRT-PCR array analysis of chemokine and receptor expression in
tumor- and thymus-derived Tregs. Gray bar indicates cluster of
genes upregulated in tumor-derived Tregs; receptor gene names
are listed. (E) Interaction network analysis of chemokine ligand
gene expression detected in SCC cells (circles; left) and
corresponding receptor gene expression detected in Tregs
(squares; right). Genes are ordered vertically by fold change. Light
gray lines connect receptor–ligand pairs; green lines indicate pairs
upregulated at least two-fold in SCC FAK-wt cells and tumorderived Tregs. (F) qRT-PCR analysis of selected cytokine and
chemokine gene expression in SCC cells. ****P < 0.0001 (Sidakcorrected one-way ANOVA). Data are represented as mean ± SEM.#
GAPDH!
!
T!
D
!
-W K-/- -K
K
A
A
A
F
F
F
C C
C
SC SC SC
0.0
H!
FAK!
K
SCC FAK-NLS
400
GAPDH!
PARP!
Nucleus!
Volume (mm3)
Rel. Intensity
***!
FAK!
G!
SCC FAK-wt pLKO
SCC FAK-/-
***!
PARP!
Cytoplasm!
Ccl5!
1.0
GAPDH!
Nucleus!
No. of Tregs / mg of tumor
10 14 17 21 24 27
Days
FAK!
20
FA
SC FA
K
C K-w
FA wt
t
S
K sh C pL
-w R C K
t s N FA O
hR A-C K
N c l -/A 5
-C P
cl 1
5
P2
7
SC
3
40
SC
C
0
!
T!
LS
!
-W K-/- -N
K
FA FA
FA
C C
C
SC SC SC
K
C
Rel. Intensity
SC
C
C
FA
SC FA
K
C K-w
FA wt
t
S
K sh C pLK
-w R C
t s N FA O
hR A-C K
N c l -/A 5
-C P
cl 1
5
P2
100
C
SC FAK
S C C -w
C FA t
FA K
K -/-N
LS
CD8+ : Treg Ratio
%CD4+
%CD45+
Ly6Clo!
Ly6Chi!
300
200
D!
60
Figure 6. Nuclear FAK interacts with regulators of Ccl5
transcription. (A) Sucrose fractionation of soluble chromatin
preparation from nuclei isolated from SCC FAK-wt cells. Protein
preparations recovered from each fraction were analyzed by
western blotting with anti-FAK, HP1α, and histone H3 antibodies
(top three panels). DNA recovered from each fraction was run down
an agarose gel alongside a 1 kilobase (kb) and 100 base pair (bp)
ladder (bottom panel). Fraction 7 (black arrowhead) represents the
chromatin-containing fraction. (B) Schematic detailing the workflow
used for proteomic analysis of the nuclear FAK interactome in the
context of Ccl5 transcription factors (TFs). (C) Interaction network
analysis of proteins that bind FAK in the nucleus of SCC cells.
Predicted Ccl5 TFs (squares; bottom) and respective TF binders
(circles; top) enriched by at least four-fold in nuclear FAK
immunoprecipitations (SCC FAK-wt over SCC FAK-/- controls; p <
0.05) are shown (stringent network). Ccl5 TFs not detected (ND)
are shown as gray squares. TF complexes or groups are indicated;
proteins are labeled with gene names for clarity. TF binders are
aligned above TF groups with which there are the greatest number
of reported interactions. For full network, see Supplementary Figure
8. For full protein interaction list, see Supplementary Table 1. (D)
Isolation of the TFIID component TAF9 by FAK immunoprecipitation
(IP) from SCC FAK-wt cell nuclear extracts. #
Figure 6. Key points:
•  FAK is associated with chromatin, and exists in complex with transcription factors and transcriptional
regulators that are linked to regulation of Ccl5 expression.
•  FAK interacts with core transcriptional machinery to influence gene transcription and promote tumor
immune escape.
A!
B!
400
SCC FAK-wt Vehicle Treated
SCC FAK-/- Vehicle Treated
SCC FAK-wt VS-4718 Treated
SCC FAK-/- VS-4718 Treated
300
CD4+ FoxP3+ CD25+
30 SCC FAK-wt
****
SCC FAK-/-
***
***
20
%CD4+
21
300
0
CD4+CD25+FoxP3+!
200
10
100
0
Ve
hi
cl
e
VS
-4
71
8
Ve
hi
cl
e
VS
-4
71
8
17
0.0
400
0
0
C!
3
SCC FAK-wt
SCC FAK-wt
SCC FAK-wt
SCC FAK-wt
1000
7
10 14
Day
17
24
D! 400
Isotype Ctrl + Vehicle
CD8+ Vehicle
Isotype Ctrl +VS-4718
CD8+ VS-4718
800
600
400
200
0
21
Volume (mm3)
10
14
Days
0.2
0.5
E!
Volume (mm3)
7
0
0.4
F i g u r e 3 . FA K r e g u l a t e s t h e i m m u n o - s u p p r e s s i v e
microenvironment. (A) FACS quantification of Ly6Chi and Ly6Clow
macrophage populations expressed as a percentage of tumor
infiltrating CD45 + leukocytes. (B) FACS quantification of
Ly6ChiGr1low (M-MDSC) and Ly6CintGr1hi (G-MDSC) populations
expressed as a percentage of tumor infiltrating CD45+ leukocytes.
(C) Quantification of CD4+CD25+FoxP3+ Tregs expressed as a
percentage of tumor infiltrating CD4+ T-cells. (D) CD8+ T-cell to
Treg ratio calculated using mean values from Figs. 2D and 3C. (E)
SCC FAK-wt tumor growth in FVB mice treated with anti-CD25
clone PC61.5 T-cell depleting antibodies. Values represent the
mean ± SEM from 6 tumors. (F) FACS analysis of Helios
expression in CD4+CD25+FOXP3+ tumor infiltrating Tregs and
Tregs isolated from the thymus of tumor bearing mice. Control
represents background signal from a sample stained with CD4,
CD25, and FoxP3 conjugated antibodies but not Helios.
Representative replicates are shown in different colors for thymus
and SCC FAK-wt samples. Data are represented as mean ± SEM
unless stated otherwise. FACS analysis represents the mean from
N = 5. p-value = Not significant >0.05, * <0.05, ** <0.01, ***
<0.001, **** <0.0001.!
500
0.0
Tumour Volume (mm3)
C!
3
0.6
C
SC FAK
S C C F -wt
C AK
F A -/K
-k
d
0
17
10
0
D!
SC
14
15
-w
S C FA t
C K-/
FA K
-k
d
10
Days
20
SC
7
5
CD4+ FoxP3+ CD25+
25
**** ****
SC
3
****
C
SC FA
K
S C C F -w
C AK t
F SC A K /-k
C
d
SC FA
K
C
S C F -w
C AK t
F A -/
K -k
d
0
*
SC
0
0
G-MDSC
10
0
K
-w
FA t
K
C
F A -/K
-k
d
100
M-MDSC
C!
5
C
200
15
CD45+ CD11b+ F4/8015
SC
*
*
200
30
SC
****
400
****
300
45
%CD45+
Volume (mm3)
Volume (mm3)
600
SCC FAK-wt
SCC FAK-/-
B!
CD45+ CD11b+ F4/80hi
60
FA
400
SCC FAK-wt
SCC FAK-/-
A!
C
800
FVB (Immune-competent)!
SC
B!
CD1-Nude (Immune-deficient)!
Volume (mm3)
A!
Figure 1. Loss of FAK or FAK kinase activity results in
CD8+ T-cell dependent SCC tumor clearance. (A) SCC
FAK-wt and SCC FAK-/- subcutaneous tumor growth in
immune-deficient CD-1 nude mice. *p<0.05, ****p<0.0001
(Sidak corrected 2way ANOVA). (B) SCC FAK-wt and SCC
FAK-/- subcutaneous tumor growth in immune-competent
FVB mice. *p<0.05, ****p<0.0001 (Sidak corrected 2way
ANOVA). (C) SCC FAK-/- tumor growth in FVB mice treated
with T-cell depleting antibodies. p<0.0001 (Turkey corrected
2way ANOVA, comparison to SCC FAK-/-). (D) SCC FAK-wt
tumor growth in FVB mice treated with T-cell depleting
antibodies. p<0.0001 (Turkey corrected 2way ANOVA,
comparison to SCC FAK-wt). (E) Top - Secondary tumor rechallenge with SCC FAK-/- cells following a pre-challenge
with SCC FAK-/- cells and a 7 day tumor free period. Middle
- Secondary tumor re-challenge with SCC FAK-wt cells
following a pre-challenge with SCC FAK-/- cells and a 7 day
tumor free period. Bottom – Subcutaneous growth of SCC
FAK-wt and SCC FAK-/- tumors injected at 28 into a cohort
of mice in which no pre-challenge had been performed. (F)
Tumor growth measurements in FVB mice following
subcutaneous injection of SCC FAK-wt, SCC FAK-/-, and
SCC FAK-kd cells. Statistics represent FAK-wt vs FAK-/-.
Data are represented as mean ± SEM, N = 5 – 6 tumors /
group. p-value = Not significant >0.05, * <0.05, ** <0.01, ***
<0.001, **** <0.0001.!
****!
****!
****!
1.0
C!
SCC FAK-wt pLKO
SCC FAK-/SCC FAK-wt shRNA-CCL5 P1
SCC FAK-wt shRNA-CCL5 P2
SC
Here we report a completely new function of the integrin effector signaling protein Focal Adhesion Kinase (FAK) in driving anti-tumor immune evasion. Specifically, the activity of nucleartargeted FAK in cancer cells drives recruitment and retention of intra-tumoral regulatory T-cells (Tregs) by transcriptionally regulating chemokine and cytokine ligand-receptor networks, crucially
including transcription of Ccl5 and Tgfb2. In turn, these changes inhibit antigen-primed cytotoxic CD8+ T-cell activity in the tumor microenvironment, permitting survival and growth of FAKexpressing tumors. We show that immune evasion requires nuclear FAK’s catalytic activity and nuclear localization. Mechanistically, nuclear FAK is associated with chromatin and exists in complex
with transcription factors and their upstream transcriptional regulators known to control Ccl5 expression. Moreover, FAK’s nuclear immune-modulatory activities are likely specific to cancer
cells, as normal keratinocyte counterparts of SCC cells do not have nuclear FAK. Finally, we show that a small molecule FAK kinase inhibitor, VS-4718, which is currently in clinical development,
drives depletion of Tregs and promotes CD8+ T-cell-mediated tumor clearance. It is therefore likely that FAK inhibitors may trigger immune-mediated tumor regression and provide previously
unrecognized therapeutic benefit.
B!
Ccl5!
Volume (mm3)
Introduction
A!
SCC FAK-wt Vehicle Treated
SCC FAK-/- Vehicle Treated
SCC FAK-wt VS-4718 Treated
SCC FAK-/- VS-4718 Treated
300
200
Start Treatment
100
0
0
3
7
10 14
Days
17
21
0
3
7 10 14 17 21 24 28 31
Days
Figure 7. The FAK kinase inhibitor VS-4718 leads to immune
mediated SCC clearance. (A) SCC FAK-wt and SCC FAK-/- tumor
growth in FVB animals treated with either vehicle alone or
VS-4718. Treatment started 24hrs pre-tumor cell inoculation and
continued for the duration of the experiment. N = 6 tumors / group.
(B) FACS analysis of tumor infiltrating CD4+CD25+FoxP3+ Tregs
expressed as a percentage of tumor infiltrating CD4+ T-cells.
***p<0.001, ****p<0.0001 (Tukey-corrected one-way ANOVA). (C)
SCC FAK-wt tumor growth in FVB animals treated with either
vehicle or VS-4718 and either isotype control or CD8 depleting
antibodies. N = 6 tumors / group. (D) SCC FAK-wt and SCC FAK-/tumor growth in FVB animals treated with either vehicle or
VS-4718. Treatment started 5 days post-tumor cell inoculation
(grey dashed arrow / line) and continued for the duration of the
experiment. N = 6 tumors / group. Data are represented as mean ±
SEM. !
!
Figure 7. Key points:
•  VS-4718 treatment drives immune-mediated regression of SCC FAK-wt tumors.
•  VS-4718 modifies the immuno-suppressive tumor environment to promote tumor regression.
•  FAK inhibitors may trigger immune-mediated tumor regression and provide previously unrecognized
therapeutic benefit.
Email [email protected] www.ecrc.ed.ac.uk www.igmm.ac.uk