Circulating tumor cells (CTCs) from breast cancer patients were cultured to form mammospheres which exhibit stem-like properties. The mammospheres and total CTCs were tested for chemosensitivity to various drugs. Mammospheres were found to be significantly more chemoresistant than total CTCs. Salinomycin and curcumin showed high efficacy against mammospheres, with salinomycin destroying nearly all cells. The results demonstrate that stem-like CTCs circulating in blood are more resistant to chemotherapy than other CTCs, and salinomycin is a promising therapeutic for targeting chemoresistant tumor cells.

1. Maintrac®
In vitro chemosensitivity testing of mammospheres
cultured from circulating epithelial tumor cells (CETCs)
of breast cancer patients.
Comparison to chemosensitivity of total CETCs
Based on the poster of M. Pizon, D.Zimon, U. Pachmann, K. Pachmann
Transfusion Medicine Center Bayreuth TZB, Germany

2. Circulating Tumor Cells
from solid tumors
S Solid tumors are from
epithelial origin
S Solid tumors dissiminate
epithelial cells
⇒ CETCs
(circulating epithelial
tumor cells)
Y.
Shiozawa,
A
M
Havens,
K
J
Pienta
and
R
S
Taichman,
Leukemia
(2008)
22,
941–950

3. Background
S In vitro chemosensitivity testing of
circulating epithelial tumor cells
(CETCs) provides real-time
information about the sensitivity of
the tumor cells present in the patient
and correlates with treatment
success. Nevertheless, a fraction of
CETCs can survive after
conventional chemotherapy and
grow into distant metastasis.
This may be a subpopulation of
CETCs with proliferation activity
which has the ability to form floating
spheres in suspension culture.
Spheroids exhibit stem cell-like
properties and may be responsible for
chemo therapeutic resistance.
Therefore, the aim of our study was
to determine the efficacy of chemo
therapeutics on spheroids cultured
from CETCs.

4. Method
S The enumeration of CETCs from
patients with solid tumors in clinical
stage 1-4 was performed using the
maintrac® method. Subsequently,
CETCs in the context of the
surrounding white blood cells were
cultured in a suspension culture
allowing for spheroid formation.
To evaluate the cytotoxic effect of
drugs on CETCs and spheroids we
exposed them to anticancer drugs in
short time culture in different
concentrations and for different
periods of time.

5. Maintrac liquid biopsy
cell staining allows
quantitative detection
of vital circulating
tumor cells
NO fixation.
NO isolation.
NO extraction.
Fluorochrome
labeled antibody
FITC
Circulating
tumor cell
Surface
antigen
HEA

6. • 1 ml EDTA Blood
• Lysis of red blood cells
• One centrifugation step
Culture of all white
blood cells under
conditions favoring
growth of epithelial cells
and determination of
sphere formation at
different times of culture

7. • Addition of anticancer
drugs in different
concentrations
• Short time culture of
CETCs and spheroids
• Image analysis of
CETCs and spheroids
using the Scan^R
Fluorescence
microscopy

8. Results
S In contrast to CETCs, spheroids were
significantly more chemoresistant.
Active drugs led to disintegration of
tumor spheres with destruction of
part of the cells. Interestingly, some
cells in the spheres were able to
survive. Epirubicin and especially
salinomycin, a polyether ionophore
antibiotic isolated from Streptomyces
albus, showed high efficacy in a high
proportion of cells.
Furthermore, our data suggested that
curcumin, a natural biologically
active compound that is extracted
from the plant Curcuma longa is a
promising agent for cancer treatment.
Docetaxel, cyclophosphamide and
5-fluoruracil showed lower cytotoxic
effects onto the cells in the spheres.

9. Remaining live CETCs after
cytotoxic drug treatment. These
cells have an intact morphology
with a well preserved membrane
without PI staining in the nucleus.
Dead CETCs after short time
culture with cytotoxic drugs. Cells
show a positive PI staining because
of loss of membrane integrity.

10. Comparison of the cytotoxic
effect of different drugs on CETCs
and tumorspheres.
Spheroids are more resistant than
CETCs from the same patient,
which confirmed our hypothesis
that a small fraction of CETCs
has stem cell properties.
Most of the tested anticancer
therapeutics show statistically
significant higher activity on
CETCs.
Interestingly, salinomycin
treatment significantly reduces
the number of viable
tumorspheres more than of the
CETCs.

11. Examples of tumorspheres
with chemoresistance to
• cyclophosphamide,
• 5-fluorouracil,
• paclitaxel and
• docetaxel.
Tumorspheres remain alive during
short time culture (0-9h).
Compared to...

12. Tumorspheres sensitive to
• carboplatin,
• epirubicin,
• salinomycin and
• curcumin.
Carboplatin and epirubicin lead to
disintegration of tumorspheres with
destruction of part of the cells in the
spheroids.
The strong cytotoxic effect of
salinomycin is already observed at
the first point of measurement with
almost total destruction of all cells.
Curcumin works by inducing cell
death in all cells of the tumorspheres
leading to nuclear staining with
propidium iodide.

13. Conclusion
S Our results show, for the
first time that stem cells
circulating in peripheral
blood, capable of forming
spheroids are way more
resistant to anticancer
drugs than the remnant
circulating tumor cells.
We, furthermore,
demonstrate that
salinomycin efficiently
can destroy spheroids
cultured from CETCs,
strengthening its role as a
promising anti-cancer
therapeutic.

14. Thanks
for your attention...

15. Association Transfusion Medicine Center in Bayreuth - TZB
SIMFO Specialized Immunology Science + Development GmbH &
Laboratory Dr. Ulrich Pachmann
Kurpromenade 2
95448 Bayreuth
Germany
www.maintrac.com