Abstract Purification of CD10 (fibroblasts) and Epcam+ (tumor) cells

Acknowledgments
FNRS, MEDIC Foundation,
Breast Cancer Research Foundation
(BCRF, Evelyne Lauder)
Abstract
Background: Epithelial-stromal interactions are known to be important in normal mammary gland
development and to play a role in breast carcinogenesis. The aim of this study was to explore the
influence of breast tumor microenvironment on primary tumor growth, breast cancer sub-typing
and prognosis.
Methods: Myo-fibroblast cells (CD10) were isolated and purified from breast tumor (N= 28) and
normal (N=4) tissues. Gene expression analysis was performed using the Affymetrix GeneChip®
Human Genome U133 Plus 2.0 arrays. Survival analysis was carried out using 12 publicly available
microarray datasets including more than 1200 systemically untreated breast cancer patients.
Results: Breast tumor myo-fibroblast stroma cells showed an altered gene expression patterns
to the ones isolated from normal breast tissues. While some of the differentially expressed genes
are found to be associated with extracellular matrix formation/degradation and angiogenesis, the
function of several other genes remains largely unknown. Unsupervised hierarchical clustering
analysis clustered breast tumor myo-fibroblast cells into subgroups recapitulating the molecular
portraits of breast cancer based on ER, HER2 status and tumor differentiation. A stroma gene
expression signature developed from myo-fibroblast cells isolated from normal versus BC tissues
showed a statistically significant association with clinical outcome. Breast tumors with high
expression levels of the stroma signature were significantly associated with worse prognosis (HR
1.55; CI 1.20-1.99; p=5.57 10-4). This association was only observed within the clinically high risk
HER2+ subtypes. Interestingly, HER2+ tumors with high and low expression levels of the stroma
signature showed 45% and 85% distant metastasis free survival at 5-year follow-up respectively
(HR 2.53; CI 1.31-4.90; p=5.29 10-3).
Conclusions: Our results highlight the importance of tumor epithelial-stroma cell interactions in
breast carcinogenesis and breast cancer sub-typing. Moreover, it shows the role of stroma cells in
tumor dissemination particularly within the HER2+ subtype and provide basis for the development
of novel therapeutic strategies.
Experimental Protocol
Primary
tumor
Dissection
Digestion
Single cell
suspenssion
Isolation/Separation
Cancer cells
(Epcam+)
“Stromal cells”Leukocytes
(CD45+)
Epcam
CD45
CD10
Fibroblasts
(CD10+)
Mechanic
dissection Enzymatic
digestion Amplification protocol
Purification of
CD10 (fibroblasts)
and Epcam+ (tumor) cells
10
010 110 210 310 4
Epcam (c ancer cells)
Leukocytes
10
010 110 210310 4 CD10 (Fibroblasts)
Leukocytes
10010110
210 310 4
Leukocytes
10
010 110 210 3104
Leukocytes
10
010110 210 3104
Leukocytes
fibroblasts
Epcam (cancer cells)
Epcam (cancer cells)
CD10 (F ibroblasts)
51 Tumor samples
Epcam+
(Tumor cells)
N=21
CD10+
(fibroblasts)
N=28
Tumor Fibroblast Signature
CD10+ Tumor vs CD10+ Normal
Normal
CD10+
Tumor
CD10+
vs
103 probe sets
Filtre: Fold Chance >= 4 & FDR < 0.01
89 mapped
Known function
N=51 genes
Uknown function
N=38 genes
Ingenuity Pathway
Analysis
Network 1
Cancer
Cellular movement
Connective tissue disorder
Clinical Relevance?
All patients N!1400
Systemically Untreated
Unsupervised Analysis
of CD10+ and Epcam+ cells
Epcam+
(Tumor cells)
CD10+
(Fibroblasts)
Differences in Gene Expression
of CD10 (fibroblasts)
According to Molecular Subtypes
Type 1: ER-/HER2- (basal-like)
Type 2: HER2+ (HER2-like)
Type 3: ER+/HER2- (luminal-like)
L:grade 1; I:grade 2; H:grade 3
HR=1.169
CI 0.913-1.497
P=0.213
Clinical Relevance?
Molecular Subtypes
ER-/HER2- ER+/HER2-
HR=3.076 CI 1.544-6.131
P=0.001
HER2+/ER-
Tumor Fibroblast Signature
Response to Therapy
HER-2
signaling
ER
signaling
Tumor
Invasion
Immune
Response
Proliferation
Module in silico
approach
High correlation
with
stroma
signature
Neoadjuvant Epirubicine
(ER- pts only, N=81)
Potential predictive value
of the tumor invasion module
and fibroblast signature
Tumor
Invasion
(Plau)
Interactions between breast cancer cells and their stromal component:
An analysis of alterations in gene expression
Christos Sotiriou, Frank El Ouriaghli, Samira Majjaj, Benjamin Haibe-Kains, Christine Desmedt, Denis Larsimont, Martine Piccart.
Translational Research and Medical Oncology Unit, Université Libre de Bruxelles (U.L.B.), Institut Jules Bordet, 121 Boulevard de Waterloo, 1000 Brussels, Belgium