Beruflich Dokumente
Kultur Dokumente
Origin(s) ? Characterization
molecular markers for prospective identification analyses of contributions to tumor initiation, progression, heterogeneity
we
Expression able to alternative splice isoforms in of normal must be of embryonic solve the problem cancer Expression of embryonic miRNAs in cancer (Nimmo and Slack, 2009) 1884. W.S. Savory Cancer IS a caricature of development (processes, cell/microenvironment)
Dannielle Engle
Jennifer Lin
Benjamin Spike
Justin La
Samantha Cheung
placode
Artist: D Engle
No Matrigel
Matrigel
E13.5
E15.5
E16
E16.5
E18.5
adult
MaSC Frequency
8.0% 7.0% 6.0% 5.0% 4.0% 3.0% 2.0% 1.0% 0.0%
CD24
CD49f
CD49f+CD24lo/-
fStromal
CD49f+CD24hi
fMaSC
aMaSC
Which cells are the stem cells? Quantitation of Putative Bipotential CK14+ CK8+ Cells
?
Double K14+ K8+ Bipotent?
Myo Luminal
K14+
K8+
K14-K8-
K14+K8-
K14-K8+
K14+K8+
fMaSCs Generate Polarized, Multi-lineage Spheres in vitro Reflecting in vivo Transplantation Efficiencies
?
9.2
8.5
Stem Cell?
Lumen (milk?)
50
Sphere Characteristics 1. Clonal a. Single cell plating b. GFP + non-GFP cells c. Methyl-cellulose 2. Polarized, Multipotent 3. Self Renewing (dissociation / replating)
(%)
1.0
0.1
0.08
0.06
50um
NO SPHERE FORMATION
50um
Methylcellulose
NO SPHERE FORMATION
Highest Expression Similarity: CD44hiCD24lo CSC, Claudin Low
Links between fMaSC gene expression profiles and human breast cancer
fMaSCPathway and Process enrichment analysis Microarray Analysis: Identifying Cancers with fMaSC signature, therapeutic targets, antigens for purification
Total Mammary Epithelium (Lin-) 1.5fold Expression in sub-populations
aMaSC (2047)
(1238) Energy metabolism Leukocyte transendothelial migr. MAPK signaling Calcium signaling Adhesion Regulation of actin cytoskeleton (87) Wnt/Hedgehog signaling Axon guidance Cancer MAPK signaling (1171) Nucleic acid metabolism/repair Leukocyte transendo. migration Metabolism Axon guidance Cancer
fStromal (3764)
(1803) Melanogenesis Cancer Axon guidance Complement cascades Wnt/Hedgehog signaling Calcium signaling pathway Toll-like receptor signaling Hematopoiesis Depression Gap junction NK cell mediated cytotoxicity
fMaSC (3450)
(1557) Cell cycle p53 signaling pathway Nucleic acid metabolism/repair Cancer Homologous recombination Amino acid turnover Metabolism PPAR signaling pathway ECM-receptor interaction Axon guidance
Basal A
HCC70 fMaSC/fSTR-Like HCC3153 fMaSC/fSTR-Like HCC1569 fMaSC/fSTR-Like SUM225CWN fMaSC-Like HCC1143 fMaSC-Like HCC1599 fMaSC-Like HCC2157 fMaSC-Like MDAMB468 fMaSC-Like HCC1937 fMaSC-Like HCC1954 fMaSC-Like SUM190PT fMaSC-Like MDAMB157 fStromal-Like HS578T fStromal-Like BT549 fStromal-Like MDAMD436 fStromal-Like SUM1315 fStromal-Like MDAMB231 fStromal-Like SUM159PT fStromal-Like HBL100 fStromal-Like HCC38 fStromal-Like SUM14 9PT fStromal-Like MDAMB435 neither MCF10A
Benjamin Spike
Neve et al. Cancer Cell 2006 Prat et al. BCR 2010
Proliferation
-prolif
-prolif
Luminal B
>1200 Samples from Ben-Porath et al, 2008 * From Lim et al. 2010
aStromal
Random
aMaSC
fMaSC
fSTR
% overlap
Benjamin Spike
fMaSC-ii overexpressed
fSTR-iv repressed
Years
Potential Molecular TargetsErbB signaling in TNBC? Illustrates the importance of Embryonic Epithelial AND Stromal Expression Pathways in adult tumorigenesis/progression Benjamin Spike
Stromal Cell
Developing Tumor
E18.5
Adult
Wnt and HH Signaling Pathways in Cancer DNA Repair (Development) Focal Adhesion P53 Signalling
DNA Repair
Metabolism
Glycolytic shift
PKM1/2 LDHA
Cellular adhesion
PDK1
fStromal Population
Transferrin IGF-2 PAI1 PLAT(TPA) Trophinin EGFR Vitronectin Tetranectin Amphiregulin EGF
Neuregulin 1 Neuregulin 2
E-selectin
L1CAM PTPR-zeta
ITGA5
fStromal/fMaSC
GFRalpha1
Benjamin Spike
<50um
50 - 100um
>100um
250
Spheres per 2000 cells from the fMaSC-enriched population
Neratinib [M]
0.001
0.01
0.1
K14 K8
50um
K14+
K8+ K8+
K8+ K14+
K14+
FACS
K8+
K14+
K14+ K8+
K14-_ K8-
Deconvolving heterogeneity to to identify the fMaSC: Fluidigm High-throughput single cell qRT-PCR
96 wells for Single Cells
Keratin +
Keratin-
Transplantation:
+++
K14 K8 DAPI
Dannielle Engle, Justin La
Single Cell Single Cell Microfluidic RNA-Seq RT-PCR (SOLiD) (Fluidigm) Dannie Engle Justin La Ben Spike Roger Lasken (JCVI) Rashel Grindberg Joyclyn Yee
1 2 3 4 5 6 7 C
Idenitify processes and markers specific to each cell type Functional Stem Cell tests Examine cellular heterogeneity in the system Examine developmental plasticity and the flexibility of the stem cell state Identify requisite stem cell biology for application in developmental biology, regenerative medicine and Cancer therapy.
aMaSC
Differentiation
Ben Porath et al (2009) Nature Mizuno, Spike, Wahl, Levine (2010) PNAS
Acknowledgements
Dannielle Engle Jennifer Lin Benjamin Spike Samantha Cheung Justin La Jennifer Green TaNeashia Morrell Rose Rodewald Leo Li
Collaborators
Rashel Grindberg Joyclyn Yee Aleix Prat
Charles Perou
Positions Open!
Breast Cancer Research Program, Breast Cancer Research Foundation, Susan G. Komen for the Cure, G. Harold and Leila Y. Mathers Charitable Foundation, NCI