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the most common cancers worldwide. Though advances in prevention
and treatment have increased survival rates in other cancers, the 5-year
survival rate for HNSCC patients, approximately 50%, has remained
virtually unchanged for more than 30 years. In search of molecules with
potential therapeutic benefit, we have conducted a comprehensive
genomic and proteomic analysis of laser capture microdissected cancer
and stromal cells from a large HNSCC frozen tumor collection from
patients of distinct metastatic status. We observed that the expression
of Vascular Endothelial Growth Factor C (VEGF-C) is a prominent feature
in the most metastatic HNSCC lesions. VEGF-C is a member of VEGF
family of growth factors and is most strongly identified with the process
of lymphangiogenesis, or the growth and proliferation of the lymphatic
vasculature. This observation prompted us to focus on the dysregulation
of the lymphangiogenesis pathways in HNSCC. We have established
rapid orthotopic models of HNSCC invasion in the tongue and intra-vital
imaging approaches to monitor the process in live animals and, thus, are
uniquely able to address the role of VEGF-C in the HNSCC metastasis.
Using a lentiviral delivery system, we used two unique sequences of
shRNA against VEGF-C to knockdown its release in OSCC3, a wellcharacterized
cell line of high metastatic potential. We then injected
these cells into SCID/Nod mice using our validated model of HNSCC
metastasis, observing differences in tumor volume, metastasis and
vasculature density. In parallel, we characterized the effects of VEGF-C
secreted by OSCC3 control and VEGF-C knockdown cells on the ability
of lymphatic endothelial cells to proliferate and migrate. We aim to
demonstrate that the release of VEGF-C by tumor cells is important for
the process of lymphatic metastasis and VEGF-C signaling should be
further investigated as a therapeutic target in HNSCC.
S028
SIGNIFICANCE OF CIRCULATING TUMOR CELLS IN PATIENTS
WITH SQUAMOUS CELL CARCINOMA OF THE HEAD AND NECK.
Kris R Jatana, MD, Priya Balasubramanian, MS, Jas C Lang, PhD, Liying
Yang, PhD, Courtney A Jatana, DDS, Elisabeth White, BA, David E
Schuller, MD, Theodores N Teknos, MD, Amit Agrawal, MD, Enver Ozer,
MD, Jeffrey J Chalmers, PhD; The Ohio State University, Arthur G. James
Cancer Hospital and Solove Research Institute.
Objective: 1) To present and discuss a high performance negative
depletion methodology for the isolation of circulating tumor cells (CTCs)
in the blood of patients with squamous cell carcinoma of the head and
neck (SCCHN). 2) To begin to determine the correlation between the
presence of CTCs and clinical outcome in SCCHN patients. Design:
Prospective clinical follow-up study of SCCHN patients undergoing
surgical intervention, who had peripheral blood tested for the presence
CTCs using a negative depletion technique. Subjects: 30 patients
diagnosed with SCCHN. Intervention: A negative depletion process to
isolate and quantify CTCs from the blood of patients with SCCHN, using
immunomagnetic separation was developed and validated. To date,
this technique was performed on over 100 blood samples taken from
patients at the time of surgery, and these patients have been followed
in a prospective manner. Immunostaining for cytokeratin was performed
on the enriched samples to determine the number of CTCs extracted
from each patient’s blood sample. In addition, RNA was extracted from
these enriched samples, and RT-PCR was used to determine if mRNA
from EGFR was present. Correlation of CTCs, tumor stage, nodal
status, EGFR status, and clinical outcome was made. Main Outcome
Measure: Disease-free survival. Results: For the initial 30 patients, our
data suggests that SCCHN patients with no detectable CTCs per mL
of blood had a statistically significant higher probability of disease-free
survival (p=0.03, mean follow-up=18 months). No CTCs were found in
healthy human control blood samples. EGFR expression was present in
62% of SCCHN patients with identifiable CTCs. In addition to cytokeratin
markers, ongoing studies focus on using multicolor Confocal microscopy
investigate the expression of other markers including “cancer stem
cell” markers. Conclusions: An enrichment technology, based on
the removal of normal cells, has been used on the peripheral blood of
SCCHN patients for which outcome data is being collected. The data at
this point indicates that this detection technology is potentially sensitive
and specific enough to predict that if no CTCs were present, SCCHN
patients had improved disease-free survival. A blood test with such
a prognostic capability could have important implications in the
management of SCCHN.
www.ahns.info
S029
ENHANCED INVASIVE AND METASTATIC POTENTIAL OF CANCER
STEM CELLS IN HEAD AND NECK SQUAMOUS CELL CARCINOMA.
Samantha J Davis, BS, Vasu Divi, MD, John H Owen, BA, Silvana
Papagerakis, MD PhD, Carol R Bradford, MD, Thomas E Carey, PhD,
Mark E Prince, MD; Department of Otorhinolaryngology, University of
Michigan, Ann Arbor, MI; Massachusetts Eye and Ear Infirmary/Harvard
Medical School, Boston, MA.
Objectives: Subpopulations of highly tumorigenic cells, or cancer stem
cells (CSCs), have been identified within tumors of many types. These
cells have the unique capacity to self-renew and produce differentiated
progeny. In head and neck squamous cell carcinomas (HNSCC), the
CSC population is contained within the cell fraction that expresses
high levels of the surface molecule CD44. Although HNSCC CSCs are
known to exhibit increased tumorigenicity compared to non-CSCs,
it is unknown what role they may play in local invasion and distant
metastasis. To explore this, we have designed both in vitro and in vivo
models to study the behavior of this unique tumor cell subpopulation.
Methods: In all experiments, cells were labeled with anti-CD44
monoclonal antibody and sorted for CD44+ and CD44- fractions using
flow cytometry. For in vitro studies, cells from three HNSCC cell lines
(UMSCC-12, -14A, and -47) were serum-starved in media containing 1%
FBS for 18 hours prior to sorting. The CD44+ and CD44- populations
were plated in the upper chambers of Matrigel-coated and control
inserts, with media containing 10% FBS and 30 ng/ml EGF in the lower
chamber. Matrigel is a reconstituted basement membrane-like material
and is well described as a tool for studying invasion. The chambers were
incubated for 24 hours at 37°C. Cells remaining in the upper chamber
were removed, and cells that had migrated to the lower chamber were
fixed and stained. Cells were then counted using light microscopy.
Results are expressed as percent invasion, calculated by dividing the
number of cells that invaded through the Matrigel by the number of
cells that migrated through the corresponding control insert. For in vivo
study of CSC metastatic potential, CD44+ and CD44- cells from one
primary tumor (HN-111) and one cell line (UMSCC-6) were injected into
the tail veins of NOD/SCID mice. Lungs were harvested at six months
to evaluate for the presence of metastasis. Results: In all three HNSCC
cell lines, CD44+ cells were more invasive than CD44- cells. The increase
in invasiveness ranged from 1.2 to 2.1-fold. UMSCC-12, a laryngeal SCC
cell line, showed the greatest difference in invasion between the two
populations. UMSCC-47, an HPV-positive tongue SCC cell line, showed
an intermediate increase, while the floor-of-mouth SCC cell line UMSCC-
14A showed the smallest increase in percent invasion (see Table 1).
Three out of four mice injected with CD44+ cells and 0/4 mice injected
with CD44- cells formed lung metastasis. Two of the metastasis arose
from primary tumor CSCs injections and one from HNSCC cell line CSCs
injections (see Table 2). Conclusions: An in vitro model of invasion
and an in vivo model of metastasis to compare behavior of CD44+ and
CD44- cells support the hypothesis that CSCs have the unique capacity
for progression of disease outside the primary tumor bed. Interestingly,
CD44+ cells not only have greater ability to invade through reconstituted
basement membrane, but they also migrate more efficiently through the
control inserts. This phenomenon could be due to enhanced general
mobility of CSCs compared to the general tumor population.
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