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J Cancer Metastasis Treat 2016;2 Suppl 1

strategies. In order to monitor the effect of test has been linked to pathways that regulate stem cell self-
compounds on cancer stem cells, a panel of markers, renewal and pluripotency, suggesting a new mechanism
preferably easy-to-measure surface markers, must whereby HIF proteins may drive tumor growth, through
be defined. This is, however, cumbersome, since for the generation of tumour-initiating cells or cancer
many tumor indications that marker panel is not clearly stem cells. Therefore, targeting the HIF pathway may
defined, with often non-overlapping combinations of provide a novel therapeutic avenue to target cancer
markers defining cell populations with cancer stem stem cells. We demonstrate that interfering with HIF
cell activities or tumor initiation ability. This is most pathway activation prevents mammosphere formation,
likely reflecting the changing nature of the stemness validated through indenpendent confirmation through
capacity in tumor cells. Sox2 promoter activation, Aldefluor assay and in vivo
®
proof-of concept experiments targeting tumor initiation.
Thus, the question arises as: how do we test
compounds for anti-cancer stem cell activity? The The assays performed in this work are part of StemTek
answer is: phenotypic screening. There are indeed Therapeutics portfolio of research services, specialized
several functional assays well validated in the scientific in targeting cancer stem cells for drug discovery.
literature that have been used for years associated to
the ability of cancer cells to demonstrate stem cell Key words:
behavior. The most relevant is the 3D tumor spheroid Cancer stem cells, 3D spheroids, drug development
assay. This assay has been used to uncover and culture
stem cells from many tissues as well as from tumors. A23
There are multiple reports now that show that spheroid CD34+ cells in blood and primary tumor foci
derived cells are enriched in tumor initiating or cancer in head and neck squamous cell carcinoma
stem cells, derived from cell lines and from natural
fresh tumors as well. There are several conceptual patients receiving radiotherapy
considerations that need to be taken into account in Elena Selivanova, Vyacheslav Andreev, Sergey
order to apply this assay to cancer stem cells:
Makarenko, Irina Zamulaeva
1. This assay may be used to assess stem cell content A. Tsyb Medical Radiological Research Centre - branch of the National
only when 3D spheroids are formed through cell growth Medical Research Radiological Centre of the Ministry of Health of the
from single cells, not by aggregating cells. Aggregation Russian Federation, Obninsk, Russia
does not impose the growth restriction necessary to
allow stem cells to differentially survive. The aim of this work was to elucidate the regularities
of changes in the frequency of CD34+ hematopoietic
2. Not every tumor and cell line is amenable to spheroid stem cells (HSCs) circulating in peripheral blood and
growth. This is related to cell adhesion expression accumulating in primary tumor focus in patients with
profile, as some cells will not form spheroids but head and neck squamous cell carcinoma (HNSCC)
disperse cell clusters. Therefore only validated cell after their exposure to the low-LET ionizing radiation
lines may be used on the spheroid assay. receiving radiotherapy.

3. Care must be taken with cytotoxic compounds. A A study group consisted of 35 patients with HNSCC in
non-specific cell toxic drug will kill cells regardless of age from 44 to 85 years (mean age 59.5 ± 1.3 years).
culture conditions, thus only non-toxic concentrations The frequency of HSCs were determined in patients’
may be used. biopsies and blood cell samples before treatment and
at 24 h after receiving of the subsequent local dose of
The tumor spheroid assay can measure stem cell γ-irradiation of 10 Gy with conventionally fractionated
number in the parental population, by looking at the dose of 2 Gy daily. Stem cell viability and a number of
number of spheres obtained or sphere forming cell HCS were evaluated in the disrupted and dissociated
frequency through limiting dilution assay. But it can tumor pieces and in blood cell samples by three-color
also measure effect on stem cell proliferation by FACS analysis using DNA binding dye Hoechst33342
measuring sphere diameter upon time. and anti-CD45 and CD34 antibodies.

Here we describe the use of 3D spheroid models The high individual variability in the frequency of
to profile compound activity against cancer stem CD45lowCD34+HSCs was found in the tumor biopsies
cells. Furthermore, a case of compounds preventing and blood obtained from patients before treatment. The
hypoxia-inducible transcription factor (HIFs) activity is median frequency of CD34+ cells before treatment was
presented. Recently, HIF transcription factor biology in the blood of 4.9.10-4, in biopsy material is 1.6.10-2.
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Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ November 16, 2016