chemosensitivity on circulating tumor spheres

S

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

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  

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.

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.

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

• 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

• 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

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.

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.

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.

Examples of tumorspheres with chemoresistance to • cyclophosphamide, • 5-fluorouracil, • paclitaxel and • docetaxel.

Tumorspheres remain alive during short time culture (0-9h).

Compared to...

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.

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.

S

Thanks for your attention...

Association Transfusion Medicine Center in Bayreuth - TZB

SIMFO Specialized Immunology Science + Development GmbH & Laboratory Dr. Ulrich Pachmann

Kurpromenade 295448 Bayreuth Germany

www.maintrac.com