Workshop 4: Tumor Heterogeneity and the Microenvironment

(February 2,2015 - February 6,2015 )

Organizers


Alexander Anderson
Integrated Mathematical Oncology, H. Lee Moffitt Cancer Center & Research Institute
Trevor Graham
Tumour Biology, Barts Cancer Institute, QMUL
Michael Ostrowski
Ohio State Comprehensive Cancer Center, Ohio State University
Charlie Swanton
London Research Institute

Heterogeneity in cancer is an observed fact, both genetically and phenotypically. Cell-cell variation is seen in almost all aspects of cancer from early development through to invasion and subsequent metastasis. Our current understanding of this heterogeneity has mainly focused at the genetic scale with little information on how this variation translates to actual changes in cell phenotypic behavior. Given that many genotypes can lead to the same cellular phenotype, we must also understand the range and scope of this heterogeneity at the phenotypic scale as ultimately this variability will dictate the aggressiveness of the tumor and its treatability. Central to our understanding of this heterogeneity is how the tumor cells interact with each other and with their microenvironment.

The tumor microenvironment is not simply the extra cellular matrix, but a complex milieu consisting of growth promoting and inhibiting factors, nutrients (including oxygen and glucose), chemokines, and importantly other cell types including (but not limited to) fibroblasts, immune cells, endothelial cells and normal epithelial cells. These microenvironmental factors and different cell types interact with one another and the tumor as it grows. The role of endothelial cells and the immune system in cancer development are fairly well established, but less is known about the function of host fibroblasts in this process. Most solid tumors present as dense fibrotic masses, which suggests that fibroblasts contribute to tumor growth by infiltrating and depositing extracellular matrix proteins. In addition, the phenotype of fibroblasts found within and around tumors (activated fibroblasts or cancer associated fibroblasts: CAFs) is different to normal fibroblasts, and closely resembles myofibroblasts. Fibroblasts act in wound healing, angiogenesis and tissue remodeling by releasing growth factors and proteases such as matrix metalloproteinases. They also deposit matrix proteins such as laminin, tenascin and fibronectin. Therefore, if the growing tumor can co-opt such fibroblasts it has an unlimited source of many of the fundamental elements required for growth and invasion.

The two central themes of this workshop are:

  • Heterogeneity (be it phenotypic, signaling or genotypic), and
  • Microenvironment (ECM, nutrients, fibroblasts and immune cells).

Since a highly heterogeneous tumor has the potential to adapt to any microenvironment, understanding how interactions between the growing tumor and its microenvironment modulate tumor heterogeneity is critical to unraveling the mechanisms of cancer initiation.

Accepted Speakers

Alexander Anderson
Integrated Mathematical Oncology, H. Lee Moffitt Cancer Center & Research Institute
Fran Balkwill
Centre for Cancer and Inflammation, Queen Mary University of London
Mary Helen Barcellos-Hoff
Robert Clarke
Joseph Costello
Christina Curtis
Medicine and Genetics, Stanford University
Elza De Bruin
Research Dep of Oncology, University College London Cancer Institute
Jude Fitzgibbon
Robert Gatenby
H. Lee Moffitt Cancer Center & Research Institute
Philip Gerlee
Mathematical Sciences, Chalmers University of Technology
Trevor Graham
Tumour Biology, Barts Cancer Institute, QMUL
Simon Hayward
Surgery, NorthShore University HealthSystem
Sui Huang
Cell and Molecular Biology, Northwestern University Medical School
Shelley Hwang
Simon Leedham
Wellcome Trust Centre for Human Genetics, University of Oxford
Gustavo Leone
Molecular Virology, Immunology, and Medical Genetics, The Ohio State University
Morag Park
Biochemistry and Oncology, McGill University
Vito Quaranta
Department of Cancer Biology, Vanderbilt University
Sergio Quezada
Research department of haematology, University College London Cancer Institute
Erik Sahai
Tumor Cell Biology Lab, London Research Institute
Owen Sansom
Colorectal Cancer and Wnt Signalling, Cancer Research UK Beatson Insititue
Richard Sole
Andrea Sottoriva
Centre for Evolution and Cancer, The Institute of Cancer Research
Zoltan Szallasi
Thea Tlsty
Richard White
Gastrointestinal Oncology Service, Memorial Sloan-Kettering Cancer Center
Daniel Worthley
Medicine, University of Adelaide
Yinyin Yuan
Centre for Evolution and Cancer, The Institute of Cancer Research
Monday, February 2, 2015
Time Session
08:00 AM

Shuttle to MBI

08:15 AM
09:00 AM

Breakfast

09:00 AM
09:30 AM

Greetings and info from MBI - Marty Golubitsky

09:30 AM
10:15 AM
Mary Helen Barcellos-Hoff
10:15 AM
11:00 AM
Christina Curtis
11:00 AM
11:30 AM

Break

11:30 AM
12:15 PM
Jude Fitzgibbon - Sequential monitoring of Follicular lymphoma uncovers Rich and Sparse patterns of evolution

Follicular lymphoma is an incurable malignancy, with transformation to an aggressive subtype representing a critical event during disease progression. We performed whole-genome or whole-exome sequencing on 10 follicular lymphoma-transformed follicular lymphoma pairs followed by deep sequencing of 28 genes in an extension cohort, and we report the key events and evolutionary processes governing tumor initiation and transformation. Tumor evolution occurred through either a 'rich' or 'sparse' ancestral common progenitor clone (CPC). We identified recurrent mutations in linker histone, JAK-STAT signaling, NF-κB signaling and B cell developmental genes. Longitudinal analyses identified early driver mutations in chromatin regulator genes (CREBBP, EZH2 and KMT2D (MLL2)), whereas mutations in EBF1 and regulators of NF-κB signaling (MYD88 and TNFAIP3) were gained at transformation. Collectively, this study provides new insights into the genetic basis of follicular lymphoma and the clonal dynamics of transformation and suggests that personalizing therapies to target key genetic alterations in the CPC represents an attractive therapeutic strategy.

12:15 PM
01:00 PM
Sui Huang
01:00 PM
02:15 PM

Lunch Break

02:15 PM
03:00 PM
Joseph Costello - Under Pressure: Tumor evolution after therapy

Abstract not submitted.

03:00 PM
03:45 PM
Morag Park
03:45 PM
04:00 PM

Break

04:00 PM
04:45 PM
Robert Gatenby
04:45 PM
05:00 PM

Discussion

05:00 PM
07:00 PM

Reception and Poster Session

07:00 PM

Shuttle pick-up from MBI

Tuesday, February 3, 2015
Time Session
08:00 AM

Shuttle to MBI

08:15 AM
09:00 AM

Breakfast

09:00 AM
09:45 AM
Richard White - Assessing normal and cancer somatic variation with RAD-seq

Both normal and cancerous tissues are able to acquire genetic variation over time. The spectrum of cancer mutations has been well characterized through projects such as the TCGA, yet the degree of normal somatic variation within an individual, called somatic mosaicism, has been poorly characterized. To understand cancer phylogenies will require a deeper appreciation and understanding of normal somatic variation, but techniques such as whole-genome and exome sequencing are not efficient for the construction of such trees. To overcome this, we are using a reduced representation sequencing technique called RAD-seq to assess genetic variation across normal and cancer tissues. In this approach, DNA samples are digested with a restriction enzyme to give a desired amount of genome coverage, and then only those portions of the genome containing those cut sites undergo Illumina sequencing. We have applied the RAD-seq pipeline to a series of normal and cancer tissues from both humans (pancreatic cancer patients) as well as zebrafish (melanoma bearing animals), sequencing both the tumor itself, individual metastases, and several normal tissues such as lung and brain. We are using this data to understand the patterns of SNVs and copy number changes in each of these tissues, and across species. The application of RAD-seq will allow for high-throughput, deep coverage of genomic variation at a fraction of the cost of traditional sequencing approaches, and will be broadly applicable to experiments focusing on cancer evolution.

09:45 AM
10:30 AM
Alexander Anderson - In vivo and in vitro modeling of epithelial and stromal heterogeneity in tumors

Both stromal and epithelial cells in tumors exhibit marked heterogeneity that can affect communication within and between tissue layers resulting in alterations in cell proliferation, survival and death. Many of these interactions are cooperative rather than competitive. The various signaling axes represent a complex network of altered ligand/receptor availability that can be modified to alter growth and progression. A more complete understanding of cellular complexity in both stromal and epithelial tissues should allow us to model these intercellular communications with a view to identifying key nodes that can be coordinately modulated to restrict tumor growth.

10:30 AM
11:00 AM

Break

11:00 AM
11:45 AM
Simon Leedham - Morphogenic and microenvironmental control of intestinal stem cell fate

The rapid turnover of the intestinal epithelium is supported by tissue specific stem cells thought to be located at the base of the intestinal crypt - the basic functional unit of the gut. Stem cell progeny progressively proliferate and differentiate as they move out of the crypt base stem cell niche and along the crypt-villus axis of the intestine. This system is carefully orchestrated by interacting endogenous epithelial and paracrine secreted morphogen gradients, with cell fate coupled to, and determined by, a cells position within these gradients. It has been recently suggested that stem cell fate is similarly influenced by microenvironmental factors, and that stem cell function is not cell autonomous. Recent work has shown that cells outside of the stem cell niche can reacquire stem cell properties to assist in crypt regeneration and that disruption of mucosal morphogenic gradients can cause tumour initiation from cells that have exited the crypt base stem cell niche.

This talk will explore the exciting developments in the homeostatic and pathological microenvironmental control of intestinal stem cells and discuss the implications of this for colorectal cancer heterogeneity and chemotherapeutic strategy.

11:45 AM
12:30 PM
Daniel Worthley - Connective tissue stem cells in the tumor microenvironment

Abstract not submitted.

12:30 PM
01:45 PM

Lunch Break

01:45 PM
02:30 PM
Owen Sansom
02:30 PM
04:15 PM

Breakout Session

04:15 PM
05:00 PM
Philip Gerlee
05:00 PM

Shuttle pick-up from MBI

Wednesday, February 4, 2015
Time Session
08:00 AM

Shuttle to MBI

08:15 AM
09:00 AM

Breakfast

09:00 AM
09:45 AM
Vito Quaranta
09:45 AM
10:30 AM
Andrea Sottoriva
10:30 AM
11:00 AM

Break

11:00 AM
11:45 AM
Zoltan Szallasi
11:45 AM
12:30 PM
Parag Mallick
12:30 PM
01:45 PM

Lunch Break

01:45 PM
02:30 PM
Erik Sahai
02:30 PM
04:15 PM

Breakout Discussion

04:15 PM
05:00 PM
Robert Clarke
05:00 PM

Shuttle pick-up from MBI

Thursday, February 5, 2015
Time Session
08:00 AM

Shuttle to MBI

08:15 AM
09:00 AM

Breakfast

09:00 AM
09:45 AM
Sergio Quezada - Targeting immune-checkpoints in cancer: New mechanistic insights

The continual interplay between the immune system and cancerous cells is thought to result in the establishment of a dynamic state of equilibrium. This equilibrium depends on the balance between subsets of effector and regulatory lymphocytes. Whereas the overall mechanisms underpinning the establishment and maintenance of the intra-tumour balance between Teff and Treg cells remain unknown, in many solid cancers it is characterized by the dominant infiltration of regulatory T cells over effector T cells resulting in a low Teff/Treg ratio. Furthermore, different subtypes of regulatory cells and inhibitory molecules such as CTLA-4 tightly control the few effector T lymphocytes that manage to infiltrate the tumour. The outcome of this balance is critical to survival, and while in a few cases the equilibrium resolves in the elimination of the tumour by the immune system, in many other cases the tumour manages to escape immune control.

Remarkably, antibodies against CTLA-4, a key immune modulatory receptor expressed on T cells, efficiently modify this balance, driving effector T cell expansion and increasing the ratio of Teff/Treg within the tumour. Whilst the high Teff/Treg ratio driven by anti-CTLA-4 directly correlates with tumour destruction in mice and humans, the mechanisms underpinning this phenomenon remain unknown.

By focusing in the study of effector and regulatory tumour-reactive CD4+ T cells my group is interested in the mechanism underpinning the activity of different immune-modulatory antibodies within the tumour microenvironment, and the potential positive and negative impact that the tumour microenvironment may have in the recruitment, survival and function of different T cell subsets. In this context and using a murine model of melanoma we have recently demonstrated that both, the change in the Teff/Treg balance as well as tumour rejection, depend on the selective depletion of tumour-infiltrating Treg cells expressing high levels of surface CTLA-4. Regulatory T cell depletion is mediated by ADCC and completely depends on the expression of FcRIV on tumour infiltrating CD11b+ myeloid cells. These results reveal novel and unexpected mechanistic insight into the activity of anti-CTLA-4-based cancer immunotherapy, and illustrate the importance of specific features of the tumour microenvironment on the final outcome of antibody-based immune-modulatory therapies.

09:45 AM
10:30 AM
Fran Balkwill - The evolving tumour microenvironment of high grade serous ovarian cancer, HGSC

I will be describing our studies on the tumour microenvironment of high grade serous ovarian cancer and inflammatory cytokine targets in this cancer.

10:30 AM
11:00 AM

Break

11:00 AM
11:45 AM
Yinyin Yuan - The Ecosystem Diversity Landscape of Breast Cancer

It is increasingly recognised that the tumor microenvironment is an important determinant in cancer progression and evolution, where tumors act as complex ecosytems involving interactions between cancer cells, stromal cells and their physical environment. To study this we developed a statistical model to systematically quantify the spatial heterogeneity of the tumor ecosystem based on automated image analysis of 1,026 Hematoxylin & Eosin (H&E) stained primary breast tumors. I will discuss the clinical implication of heterogeneous tumour ecosystem, how ecosystem diversity was capable of predicting prognosis independent to known clinical and cancer heterogeneity parameters, before moving on to the bioinformatics integration of this measurement with whole-genome genomic profiling data for all of these tumours. This helped us reveal enrichment of specific copy number alterations for certain genes in this subtype, for which an RNAi screen showed an overall pattern of increased cell invasion, suggesting that tumour ecosystem heterogeneity may aid cancer progression through its interplay with specific genomic alterations. Taken together, these results support the use of statistical modelling of spatial pathological data for a quantitative understanding of ecosystem heterogeneity and provide initial evidences of the clinical implication of tumour ecosystem diversity.

11:45 AM
12:30 PM
Thea Tlsty
12:30 PM
01:45 PM

Lunch Break

01:45 PM
02:30 PM
Trevor Graham
02:30 PM
04:15 PM

Breakout Discussion

04:15 PM
05:00 PM
Simon Hayward
05:00 PM

Shuttle pick-up from MBI

Friday, February 6, 2015
Time Session
08:00 AM

Shuttle to MBI

08:15 AM
09:00 AM

Breakfast

09:00 AM
09:45 AM

TBD

09:45 AM
10:30 AM

TBD

10:30 AM
11:00 AM

Break

11:00 AM
11:45 AM
Shelley Hwang
11:45 AM
12:30 PM
Richard Sole
12:30 PM
01:15 PM
Gustavo Leone
01:15 PM

Shuttle pick-up from MBI (One back to hotel and one to airport)

Name Email Affiliation
Altrock, Philipp paltrock@jimmy.harvard.edu Program for Evolutionary Dynamics, Harvard University
Anderson, Alexander alexander.Anderson@moffitt.org Integrated Mathematical Oncology, H. Lee Moffitt Cancer Center & Research Institute
Avendano, Alex avendano.8@osu.edu Mechanical and Aerospace Engineering, The Ohio State University
Baker, Ann-Marie a.m.c.baker@qmul.ac.uk Tumour Biology, Barts Cancer Institute, Queen Mary University of London
Balkwill, Fran f.balkwill@qmul.ac.uk Centre for Cancer and Inflammation, Queen Mary University of London
Barcellos-Hoff, Mary Helen MHBarcellos-Hoff@nyumc.org
Basanta, David david@cancerevo.org Integrated Mathematical Oncology, H. Lee Moffitt Cancer Center
Botesteanu, Dana dboteste@math.umd.edu Applied Mathematics & Scientific Computing, University of Maryland
Clarke, Robert clarker@georgetown.edu
Costello, Joseph jcostello@cc.ucsf.edu
Curtis, Christina ccurtis2@stanford.edu Medicine and Genetics, Stanford University
de Bruin, Elza e.debruin@ucl.ac.uk Research Dep of Oncology, University College London Cancer Institute
Domogauer, Jason jdomogauer@gmail.com Radiology, Rutgers New Jersey Medical School
Durrett, Rick rtd@math.duke.edu Department of Mathematics, Duke University
Eljazi, Radhia roe1@hw.ac.uk Mathematical and Computer Sceince, Heriot Watt University
Fitzgibbon, Jude j.fitzgibbon@qmul.ac.uk
Gallaher, Jill jill.gallaher@moffitt.org Integrated Mathematical Oncology, Moffitt Cancer Center
Gatenby, Robert robert.gatenby@moffitt.org H. Lee Moffitt Cancer Center & Research Institute
Gerlee, Philip philipgerlee@gmail.com Mathematical Sciences, Chalmers University of Technology
Graham, Trevor t.graham@qmul.ac.uk Tumour Biology, Barts Cancer Institute, QMUL
Hayward, Simon simon.hayward@vanderbilt.edu Surgery, NorthShore University HealthSystem
Huang, Sui sui.huang@systemsbiology.org Cell and Molecular Biology, Northwestern University Medical School
Hwang, Shelley shelley.hwang@duke.edu
Irshad, Shazia shazia.irshad@well.ox.ac.uk
Jacobsen, Karly jacobsen.50@mbi.osu.edu Mathematical Biosciences Institute, The Ohio State University
Jenkins, Robert robert.jenkins@cancer.org.uk Tumour Cell Biology Laboratory, Cancer Research UK London Research Institute
Jiang, Yi yijianglanl@gmail.com Mathematics and Statistics, Georgia State University
Kan, Rebecca Pei Qi kanrebecca@gmail.com Clinical Oncology, University of Hong Kong
Kapoor, Aastha aastha_phd@iitb.ac.in Biosciences and bioengineering, Indian Institute of Technology, Bombay
Kianercy, Ardeshir akianer1@jhmi.edu Urology, Johns Hopkins Hospital
Klimov, Sergey sklimov3@gmail.com Interdisciplinarity Biology and Mathematics, Georgia State University
Konstorum, Anna akonstor@uci.edu Mathematics, University of California, Irvine
Leedham, Simon simon.leedham@cancer.org.uk Wellcome Trust Centre for Human Genetics, University of Oxford
Leone, Gustavo gustavo.leone@osumc.edu Molecular Virology, Immunology, and Medical Genetics, The Ohio State University
Lowengrub, John lowengrb@math.uci.edu Mathematics, University of California, Irvine
Mahdipour Shirayeh, Ali ali.mahdipour@gmail.com Applied Mathematics, University of Waterloo, University of Waterloo
Martinez, Pierre p.martinez@qmul.ac.uk Tumour Biology, Evolution and Cancer laboratory, Queen Mary University of London
Oberai, Assad oberaa@rpi.edu Scientific Computation Research Center, Rensselaer Polytechnic Institute
Ostrowski, Michael michael.ostrowski@osumc.edu Ohio State Comprehensive Cancer Center, Ohio State University
Owusu, Benjamin bnyowusu@uab.edu Biochemisty & Cancer Biology, University of Alabama at Birmingham & Southern Research Institute
Park, Morag morag.park@mcgill.ca Biochemistry and Oncology, McGill University
Quaranta, Vito vito.quaranta@vanderbilt.edu Department of Cancer Biology, Vanderbilt University
Quezada, Sergio s.quezada@ucl.ac.uk Research department of haematology, University College London Cancer Institute
Robertson Tessi, Mark Mark.Robertsontessi@moffitt.org Integrated Mathematical Oncology, Moffitt Cancer Center
Sahai, Erik erik.sahai@cancer.org.uk Tumor Cell Biology Lab, London Research Institute
Sansom, Owen o.sansom@beatson.gla.ac.uk Colorectal Cancer and Wnt Signalling, Cancer Research UK Beatson Insititue
Scherz-Shouval, Ruth ruthsh@wi.mit.edu Lindquist lab, Whitehead Institute for Biomedical Research
Shahriyari, Leili shahriyari.1@osu.edu Mathematical Biosciences Institute, Ohio State University
Shibata, Darryl dshibata@usc.edu Department of Pathology, University of Southern California
Shibata, Darryl dshibata@hsc.usc.edu School of Medicine/Pathology, USC Keck School of Medicine
Sole, Richard ricard.sole@upf.edu
Sottoriva, Andrea andrea.sottoriva@icr.ac.uk Centre for Evolution and Cancer, The Institute of Cancer Research
Szallasi, Zoltan zszallasi@chip.org
Tlsty, Thea thea.tlsty@ucsf.edu
Tzedakis, Georgios giwrgos.tze@gmail.com Institute of Computer Science (ICS) - Computational Medicine Laboratory (CML), Foundation for Research and Technology - Hellas (FORTH)
Walther, Viola v.walther@qmul.ac.uk Tumour Biology, Barts Cancer Institute, Queen Mary University London
White, Richard whiter@mskcc.org Gastrointestinal Oncology Service, Memorial Sloan-Kettering Cancer Center
Worthley, Daniel daniel.worthley@adelaide.edu.au Medicine, University of Adelaide
Yuan, Yinyin yinyin.yuan@icr.ac.uk Centre for Evolution and Cancer, The Institute of Cancer Research
In vivo and in vitro modeling of epithelial and stromal heterogeneity in tumors

Both stromal and epithelial cells in tumors exhibit marked heterogeneity that can affect communication within and between tissue layers resulting in alterations in cell proliferation, survival and death. Many of these interactions are cooperative rather than competitive. The various signaling axes represent a complex network of altered ligand/receptor availability that can be modified to alter growth and progression. A more complete understanding of cellular complexity in both stromal and epithelial tissues should allow us to model these intercellular communications with a view to identifying key nodes that can be coordinately modulated to restrict tumor growth.

The evolving tumour microenvironment of high grade serous ovarian cancer, HGSC

I will be describing our studies on the tumour microenvironment of high grade serous ovarian cancer and inflammatory cytokine targets in this cancer.

Under Pressure: Tumor evolution after therapy

Abstract not submitted.

Sequential monitoring of Follicular lymphoma uncovers Rich and Sparse patterns of evolution

Follicular lymphoma is an incurable malignancy, with transformation to an aggressive subtype representing a critical event during disease progression. We performed whole-genome or whole-exome sequencing on 10 follicular lymphoma-transformed follicular lymphoma pairs followed by deep sequencing of 28 genes in an extension cohort, and we report the key events and evolutionary processes governing tumor initiation and transformation. Tumor evolution occurred through either a 'rich' or 'sparse' ancestral common progenitor clone (CPC). We identified recurrent mutations in linker histone, JAK-STAT signaling, NF-κB signaling and B cell developmental genes. Longitudinal analyses identified early driver mutations in chromatin regulator genes (CREBBP, EZH2 and KMT2D (MLL2)), whereas mutations in EBF1 and regulators of NF-κB signaling (MYD88 and TNFAIP3) were gained at transformation. Collectively, this study provides new insights into the genetic basis of follicular lymphoma and the clonal dynamics of transformation and suggests that personalizing therapies to target key genetic alterations in the CPC represents an attractive therapeutic strategy.

Morphogenic and microenvironmental control of intestinal stem cell fate

The rapid turnover of the intestinal epithelium is supported by tissue specific stem cells thought to be located at the base of the intestinal crypt - the basic functional unit of the gut. Stem cell progeny progressively proliferate and differentiate as they move out of the crypt base stem cell niche and along the crypt-villus axis of the intestine. This system is carefully orchestrated by interacting endogenous epithelial and paracrine secreted morphogen gradients, with cell fate coupled to, and determined by, a cells position within these gradients. It has been recently suggested that stem cell fate is similarly influenced by microenvironmental factors, and that stem cell function is not cell autonomous. Recent work has shown that cells outside of the stem cell niche can reacquire stem cell properties to assist in crypt regeneration and that disruption of mucosal morphogenic gradients can cause tumour initiation from cells that have exited the crypt base stem cell niche.

This talk will explore the exciting developments in the homeostatic and pathological microenvironmental control of intestinal stem cells and discuss the implications of this for colorectal cancer heterogeneity and chemotherapeutic strategy.

Targeting immune-checkpoints in cancer: New mechanistic insights

The continual interplay between the immune system and cancerous cells is thought to result in the establishment of a dynamic state of equilibrium. This equilibrium depends on the balance between subsets of effector and regulatory lymphocytes. Whereas the overall mechanisms underpinning the establishment and maintenance of the intra-tumour balance between Teff and Treg cells remain unknown, in many solid cancers it is characterized by the dominant infiltration of regulatory T cells over effector T cells resulting in a low Teff/Treg ratio. Furthermore, different subtypes of regulatory cells and inhibitory molecules such as CTLA-4 tightly control the few effector T lymphocytes that manage to infiltrate the tumour. The outcome of this balance is critical to survival, and while in a few cases the equilibrium resolves in the elimination of the tumour by the immune system, in many other cases the tumour manages to escape immune control.

Remarkably, antibodies against CTLA-4, a key immune modulatory receptor expressed on T cells, efficiently modify this balance, driving effector T cell expansion and increasing the ratio of Teff/Treg within the tumour. Whilst the high Teff/Treg ratio driven by anti-CTLA-4 directly correlates with tumour destruction in mice and humans, the mechanisms underpinning this phenomenon remain unknown.

By focusing in the study of effector and regulatory tumour-reactive CD4+ T cells my group is interested in the mechanism underpinning the activity of different immune-modulatory antibodies within the tumour microenvironment, and the potential positive and negative impact that the tumour microenvironment may have in the recruitment, survival and function of different T cell subsets. In this context and using a murine model of melanoma we have recently demonstrated that both, the change in the Teff/Treg balance as well as tumour rejection, depend on the selective depletion of tumour-infiltrating Treg cells expressing high levels of surface CTLA-4. Regulatory T cell depletion is mediated by ADCC and completely depends on the expression of FcRIV on tumour infiltrating CD11b+ myeloid cells. These results reveal novel and unexpected mechanistic insight into the activity of anti-CTLA-4-based cancer immunotherapy, and illustrate the importance of specific features of the tumour microenvironment on the final outcome of antibody-based immune-modulatory therapies.

Assessing normal and cancer somatic variation with RAD-seq

Both normal and cancerous tissues are able to acquire genetic variation over time. The spectrum of cancer mutations has been well characterized through projects such as the TCGA, yet the degree of normal somatic variation within an individual, called somatic mosaicism, has been poorly characterized. To understand cancer phylogenies will require a deeper appreciation and understanding of normal somatic variation, but techniques such as whole-genome and exome sequencing are not efficient for the construction of such trees. To overcome this, we are using a reduced representation sequencing technique called RAD-seq to assess genetic variation across normal and cancer tissues. In this approach, DNA samples are digested with a restriction enzyme to give a desired amount of genome coverage, and then only those portions of the genome containing those cut sites undergo Illumina sequencing. We have applied the RAD-seq pipeline to a series of normal and cancer tissues from both humans (pancreatic cancer patients) as well as zebrafish (melanoma bearing animals), sequencing both the tumor itself, individual metastases, and several normal tissues such as lung and brain. We are using this data to understand the patterns of SNVs and copy number changes in each of these tissues, and across species. The application of RAD-seq will allow for high-throughput, deep coverage of genomic variation at a fraction of the cost of traditional sequencing approaches, and will be broadly applicable to experiments focusing on cancer evolution.

Connective tissue stem cells in the tumor microenvironment

Abstract not submitted.

The Ecosystem Diversity Landscape of Breast Cancer

It is increasingly recognised that the tumor microenvironment is an important determinant in cancer progression and evolution, where tumors act as complex ecosytems involving interactions between cancer cells, stromal cells and their physical environment. To study this we developed a statistical model to systematically quantify the spatial heterogeneity of the tumor ecosystem based on automated image analysis of 1,026 Hematoxylin & Eosin (H&E) stained primary breast tumors. I will discuss the clinical implication of heterogeneous tumour ecosystem, how ecosystem diversity was capable of predicting prognosis independent to known clinical and cancer heterogeneity parameters, before moving on to the bioinformatics integration of this measurement with whole-genome genomic profiling data for all of these tumours. This helped us reveal enrichment of specific copy number alterations for certain genes in this subtype, for which an RNAi screen showed an overall pattern of increased cell invasion, suggesting that tumour ecosystem heterogeneity may aid cancer progression through its interplay with specific genomic alterations. Taken together, these results support the use of statistical modelling of spatial pathological data for a quantitative understanding of ecosystem heterogeneity and provide initial evidences of the clinical implication of tumour ecosystem diversity.