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“Tumor Initiating Cells in Human Cutaneous
Squamous Cell Carcinoma” Jonathan Vogel, M.D.
Dermatology Branch
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Introduction
Human cutaneous squamous cell carcinomas (SCC) exhibit a heterogeneous morphology with a developmental hierarchy of proliferating and differentiating cells may be maintained by a distinct population of cancer stem cells or tumor initiating cells (TIC). In vitro tissue culture assays and in vivo animal models that can accurately recapitulate the human cancer were developed to identify and characterize TIC . Demonstrate that a small subset of human SCC cells (~1%) expressing a prominin-1 (CD133) epitope are highly enriched for TIC in human SCC
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Human Squamous Cell Carcinoma (SCC)
Squamous cell carcinomas and basal cell carcinomas represent more than 106 cases per
year, about 25% SCC
Etiology due to DNA damage secondary to sun and environmental exposure
High incidence of SCC metastasis in transplanted and immunocompromised patients
Proliferating dysplastic keratinocytes invade locally as a mass with finger-like tumor
projections invading into tissues
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SCC continue to differentiate with Ki67+ proliferating cells
located at the periphery of SCC tumor projections
Normal Skin: K5 & Involucrin Squamous cell carcinoma
Ki67+, K14 and Involucrin
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Human SCC
Isolation of single cell suspension
Separation of cells based on cell surface markers
In vitro assay In vivo tumor initiation assay
Assess for growth/tumor formation
I
II
III
Isolation and characterization of tumor initiating cells in SCC
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Human SCC form spheroid tumor cell colonies in culture
Normal Human
Keratinocytes
Bulk Human Squamous Cell Carcinoma Cell Suspension
Tissue Culture Plate with Irradiated 3T3 feeder layer Human Squamous
Cell Carcinoma
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SCC spheroid colonies could be serially passaged without increase or decrease in colony numbers noted
Cloning cylinders used to trypsinize and
passage individual spheres Whole plate trypsinized and passaged
SCC
Input
# 1°colonies
# 2°colonies
105
49
48
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In Vivo Model for Human SCC Initiation
Successful xenografts of human SCC cell suspensions required extensive “humanization” of the graft site
Establishing this in vivo assay required 140 separate human SCC samples and 155 individual mouse xenografts over a 3 year period
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Xenograft Tumor
Original Tumor
Xenograft SCC tumor growth rate and histology was similar to original SCC tumor
Poorly Differentiated Moderately Differentiated Well Differentiated
SCC1 – Poorly Differentiated
SCC2 – Moderately Differentiated
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Human SCC
Isolation of single cell suspension
Separation of cells based on cell surface markers
In vitro assay In vivo tumor initiation assay
Assess for growth/tumor formation
I
II
III
Isolation and characterization of tumor initiating cells in SCC
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Potential Cell Surface Markers for Tumor Initiating Cells
1. Previously, panels of cell surface markers were identified for human keratinocyte stem cells in hair follicles
(CD200hi24lo34lo71lo146lo)
2. The CD44 and CD24 cell surface markers have been used to isolate tumor initiating cells from fresh human cancer
specimens, including breast, head and neck, and pancreas. The glycosylated CD133 cell surface marker has also identified
tumor initiating cells in primary human cancer, including brain (medulloblastomas and glioblastomas), colon, and pancreas.
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CD133 was expressed on scattered cell clusters in the proliferating
layer of the human SCC tumor projections
CD31 (pecam)
CD133 (promin1)
Dapi
CD31 (pecam)
CD133 (promin1)
Keratin
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CD133+ cells represent a rare subset of human SCC cells Mean 0.81% +/-0.86% n=31
Iso
typ
e
Isotype
1.02%
CD45
CD
44
C
D1
33
CD
13
3
CD71
1.29%
29.33%
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Summary of Cell Surface Markers in SCC
Proliferating SCC periphery CD71-hi (CD24- and CD146-)
Differentiating inner SCC CD24-hi and CD146-hi (CD71-)
CD133-Hi
CD200+ cells are not present in SCC and CD44+ cells were CD45+
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Human SCC
Isolation of single cell suspension
Separation of cells based on certain characteristics
In vitro assay In vivo tumor initiation assay
Assess for growth/tumor formation
I
II
III
Isolation and characterization of tumor initiating cells in SCC
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CD133+ cells isolated from SCC are enriched for spheroid colony formation
CD133+ SCC Tumor Cell Colonies
Nu
mb
er
of
Co
lon
ies
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Human SCC
Isolation of single cell suspension
Separation of cells based on certain characteristics
In vitro assay In vivo tumor initiation assay
Assess for growth/tumor formation
I
II
III
Isolation and characterization of tumor initiating cells in SCC
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Tumor growth was dependent on the number of
unsorted human SCC cells xenotransplanted
82 Total Xenographs into Nude Mice
TIC frequency = 1 / 1,400,000 Total SCC cells
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Xenotransplanted CD133+ SCC cells are highly enriched for TIC
42 total xenografts from 28 different human SCC specimens
TIC frequency = 1 / 483 CD133+ cells
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The number of implanted CD133+ SCC cells can determine xenograft size
(at 12 weeks)
1 102 103 104
Number of CD133+ cells implanted
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CD133+ SCC can be serially transplanted - demonstrating the
stem cell properties of self-renewal and tumor reconstitution
Serial transplants of CD133+
from 1’ xenografts
Primary SCC Xenografts
Secondary SCC Xenografts
FACS analysis of CD133+ /
CD45- cells in primary SCC xenograft = 0.7% (n=11)
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Human CD133+ SCC cells are enriched for TIC
when serially transplanted into mice
14 total serial xenografts from 8 different human SCC specimens
TIC frequency = 1 / 863
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Xenotransplanted CD133+ SCC cells recreates the original tumor morphology
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Different SCC histological grades had equivalent rates of tumor formation
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Different SCC histological grades had equivalent percentages of CD133+ cells
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CD133+/CD45- cells were relatively quiescent
Ce
ll n
um
be
r
DNA content
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Conclusions
A discrete small sub-population (1%) of human CD133+ SCC cells are
highly enriched for tumor-initiating cells (TIC) SCC in an in vivo SCC
xenograft model.
The CD133+ cells could recapitulate the histology and hierarchy of the
original SCC tumor.
The percentages of CD133+ TIC in SCC tumors of different histological
grades were equivalent.
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Future Considerations
Future studies will focus on how normal skin developmental programs
have been altered in SCC by analyzing TIC for:
-Global gene expression profiles
-Genetic and epigenetic changes
-Stromal microenvironment or “niche” influences on TIC behavior
Other SCC tumors, such as the highly aggressive SCC in renal transplant
patients also need to be studied.
Enriched TIC also represent potentially valuable targets for therapeutic
strategies that can selectively inhibit their growth and self-renewal.
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Acknowledgments Vogel Lab
Atsushi Terunuma
Jean-Philippe
Therrien
Manabu Ohyama
Girish Patel
Renuka Pudi
Carole Yee
Cancer stem cell
interest group -
Barbara Vonderhaar
Snorri Thorgeirsson
Laboratory of
Cellular Oncology,
Michael Radonovich
Cindy Masison
John N. Brady
Laboratory of
Pathology, CCR
Michael Emmert-Buck
Dermatology branch
Mark Udey
Maria Turner
Steven Katz
Medicine Branch
William Telford
Veena Kapor
Thanks!
NCI Stuart Yuspa
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