Integrative analysis reveals mechanisms influencing the tumor immune infiltrate in colorectal cancer
Gabriela Bindea Bernhard Mlecnik
Immune infiltration predicts patient outcome in colorectal cancer (CRC)
Mechanisms?
Galon et al. Science 2006 Pages et al. NEJM, 2005 Mlecnik et al. JCO, 2011 Patient 1 (weak) Patient 2 (moderate) Patient 3 (strong)
CD3, tumor
Integrative analysis of large scale data
Mlecnik et al. BMC Genomics. 2010
Affymetrix LDA TMA TCR Repertoire FACS aCGH MSI SNP SeqData
105 pts 150 pts 648 pts 20 pts 30 pts 216 pts 606 pts 50 pts 30 pts
- in silico predictions - hypothesis generation - experimental validation => better characterization of the tumor-immune cross talk
ClueGO & CluePedia Cytoscape apps
Overview of the biological role
Comparison of the functionality of several lists of genes A comprehensive view of a pathway/process
New markers potentially Lists of associated to pathways genes Ontologies: miRNAs Gene Ontology (GO) KEGG proteins Reactome … Experimental data In silico interactions miRNA-target gene
Bindea G, Mlecnik B, et al., Bioinformatics, 2009 Bindea G, Galon J and Mlecnik B, Bioinformatics, 2013 http://apps.cytoscape.org/ Th1 and cytotoxic T cell genes/cells are important for the survival of CRC patients
Which other markers could have related functions and a good impact on survival?
In silico prediction of chemokines and adhesion molecules
Innate and adaptive ClueGO immune genes Biological functions Pro-inflamatory qPCR data Marie, Amos Cytotoxic immune response Th1 Experimental validation T cell activation Angiogensis Negative reg of CX3CL1 immune response CXCL9 Immuno Th2 CXCL10 suppressive Tumor invasion Metastasis MADCAM1 spreading VCAM1 Experimental data ICAM1
In silico prediction
Predicted chemokines may attract specific immune cell subsets at the tumor site. Mlecnik et al, Gastroenterology 2010 Chemokines (CX3CL1, CXCL9, CXCL10) expression correlate with in situ T cell subpopulations
In situ TMA data DFS
CX3CL1-Lo CX3CL1 CXCL9-Lo CXCL10-Lo
months
Flow cytometry TMA, FACS data Marie, Amos, CD3+CD8+ Matthieu, Anne CX3CL1-Hi CXCL9-Hi CXCL10-Hi tumor (blue) CD8 (brown) Mlecnik et al. Gastroenterology 2010 The tumors are infiltrated with immune cells.
Which immune cell types infiltrate the tumors and which is their impact on the patient survival?
Cell types present at the tumor microenvironment
Neutrophils Th1, Th2 Adaptive immunity Treg, Th17 Innate immunity Memory T cells Eosinophils Tgd Dendritic cells T cells Macrophages TFH cells CD8 T cells NK cells Basophils B cells Cytotoxic cells
Normal colon Blood & lymphatic vessels Epithelium & mucosa Tumor cells Tumor The quantification of 28 different immune cell subpopulations infiltrating tumors using systems biology approaches The immunome
Purified cells
Most representative markers for 28 immune subtypes were selected.
Bindea, Mlecnik, et. al, Immunity. 2013 The immunome
Example of expression level, purified cells
The immunome Expression level, purified cells
T cell subsets T follicular helper cells
Neutrophils B cells
The immunome compendium was defined based on purified cells.
Are the immunomes of colorectal cancer patients similar/different?
The immunomes of colorectal cancer patients. 81 qPCR tested markers (>150 patients) Immunome Ontology integrated in ClueGO
The analysis of immunomes of 105 CRC Marie patients (Affymetrix) Amos
Immunome (public data purified immune cells)
=> patient clusters with different risk to relapse
Bindea, Mlecnik, et. al, Immunity. 2013 The analysis of immunomes of 105 CRC Marie patients (Affymetrix) Amos
Immunome (public data purified immune cells)
qPCR data
=> patient clusters have significant different T, Cytotoxic, B and Tfh infiltrate Bindea, Mlecnik, et. al, Immunity. 2013 Gene expression and immune density correlates within colorectal tumors
Affymetrix Affymetrix vs vs Affymetrix qPCR
qPCR qPCR vs vs qPCR TMA
The tumors have a heterogeneous immune infiltrate.
How is the intratumoral immune infiltrate organized? Is there a change of the immune infiltrate during tumor progression? What is the impact on the survival of patients?
Systems biology and data integration applied to the tumor microenvironment
Evolution in time
Spread to other organs
- localization - density Cell types - coordination Cell type interrelations Localization Density Impact on survival Impact on survival
KM Curves => new visualization types for complex data S F
=>The identification of immune cells with major D impact on patient survival
Time
The mean density of the 14 TMA markers in 107 CRC patients
Bindea, Mlecnik, et. al, Immunity. 2013 The 14 TMA markers and their positive (HR<1) or negative (HR>1) impact on patient survival. T and B cells are the most prominent cells for patient survival.
Bindea, Mlecnik, et. al, Immunity. 2013 Bindea, Mlecnik, et. al, Immunity. 2013 Chemokines and cytokines are important in the tumor-immune cross-talk.
Are the chemokine & cytokine levels within tumor microenvironment affected by genetic alterations that occur in tumor cells?
Amplifications and Deletions, chemokines & cytokines Array Comparative Genomic Hybridization (aCGH) data
Amplifications Pt1 aCGH data Deletions Anna
Chromosomes
CRC patients s e n i k o t y C
Genomic alterations Del No Amp
All chemokines Amp & cytokines Expression level Del No CXCL13 and IL15 Impact on survival
Out of all chemokine & cytokine investigated in groups of patients with/without aberrations only few have significant difference in the gene expression and impact on patient survival (CXCL13 and IL15) Chemokines. The impact of CXCL13 deletion
B (CD20) Tfh (CXCR5)
TMA data Marie, Amos CT IM CT IM
Patients with CXCL13 deletion have significant decreased CXCL13 expression and increased risk to relapse. The CXCL13 deletion is a mechanism associated with B and Tfh infiltration.
Bindea, Mlecnik, et. al, Immunity. 2013 CXCL13 and IL21 within the intratumoral immune cell network (Immunome Ontology, CluePedia)
Tfh generation is mediated by IL21 (Nurieva et al., 2008) and through the IL21 pathway, Tfh cells give rise to transferable memory cells with plasticity (Luthje et al., 2012). IL21 promotes CD8 T cell activity via Tbet (Sutherland et al., 2013) Bindea, Mlecnik, et. al, Immunity. 2013 Cytokines. The impact of the IL15 deletion ) % (
n o
i No aberration s s e r p x
e Deletion
e n e G T cells
Enriched genes Proliferation data Maristella (STRING)
Pathways Enriched genes (GO) (Affy CRC data)
Enriched miRNAs (miRWalk)
Patients with IL15 deletion have significant lower IL15 expression, increased risk to relapse and decreased T cell proliferation compared to patients without aberrations.
The IL15 deletion is a mechanism associated with T and B cell proliferation.
Mlecnik, Bindea et. al, Sci Transl Med. 2014 New mechanisms explaining ClueGO, CluePedia differences in tumor Data integration immune infiltrate Systems biology In silico predictions Experimental validation
Mechanisms explaining differences in tumor immune infiltrate
1) Predicted chemokines and adhesion molecules may attract specific immune cell subsets at the tumor site.
2) CXCL13 deletion is associated with Tfh and B cell infiltration. CXCL13 and IL21 are pivotal factors of the Tfh-B cell axis.
3) IL15 deletion is a mechanism associated with the T and B cell proliferation.
Acknowledgements
Laboratory of Integrative Cancer Immunology Collaborators Jérôme Galon Anne Berger, HEGP Franck Pagès Zlatko Trajanoski, ICBI Innsbruck Bernhard Mlecnik Mihaela Angelova, ICBI Innsbruck Gabriela Bindea Michael Speicher, Med Uni Graz Marie Tosolini Anna Obenauf, Med Uni Graz Amos Kirilovsky Marc Van Den Eynde, Brussels Maria Stella Sasso Viia Valge-Archer, MedImmune Matthieu Camus Helen Angell, MedImmune Anne Coste Maximilian Waldner, Med Klinik Erlangen Sarah Church Teams 13, 11 CRC Pauline Maby Tessa Fredriksen Lucie Lafontaine Amélie Bilocq Stephanie Mauger
Integrative analysis reveals mechanisms influencing the tumor immune infiltrate in colorectal cancer
Gabriela Bindea Bernhard Mlecnik