916 - Ex-vivo drug sensitivity of primary breast cancer stems cell populations to potentiate therapeutic strategy for treatment resistant breast cancer

Background

Emergence of drug resistant primary breast tumors has remained a major concern in disease handling. Breast cancer stem cells comprise a relatively less differentiated subpopulations of breast cancer with high expression of CD44, metastatic potential and drug recalcitrant, thus imparting major global concern in tackling breast cancer. Our work describe the effect of ethanolic extract of Anthocephalus cadamba (CLE) on breast cancer stem cell populations (CD44+/CD24- and/or EpCAM+) cultured ex-vivo.

Methods

Ethanolic leaf extract of Anthocephalus cadamba was made at a concentration of 10mg/ml. Anticancer effects of this extract was compared with the effect of Gemcitabine in breast cancer stem cells isolated from primary breast tumour. Breast tumor samples (n = 20) were homogenized with collagenase-dispase solution followed by trypsinization (1), plated (1x10⁶cells/ml) in DMEM-F12 media with supplements and antibiotics. MTT assay was carried out following established protocol (2). Cells were cultured and treated with CLE or Gemicitabine at their IC-50 dosage. RNA extraction, semiquantitative RT-PCR and qRT-PCR for expression analysis of stem cell markers (SOX2 and NANOG1) was carried out following established protocol. Immunophenotyping to detect stem cell markers in both treated and untreated cells were carried out following established protocol.

Results

Our data showed Gemcitabine, and CLE reflected 50% inhibitory effect at IC-50 doses of 50mM, 50mg/ml respectively (Fig 1). In both semiquantitative and q-RT-PCR, drug treatment resulted in reduction of copy number of SOX2 and NANOG1 amplicons (of 228, and 173bp respectively). (Fig 2). Immunophenotyping data showed, treatment with CLE showed 63% reduction of both CD44⁺ EpCAM⁺ populations (Fig 3).

Conclusions

Our data showed efficacy of CLE in attenuating breast cancer stem cells.

Editorial acknowledgement

We acknowledge Indian Council of Medical Research ( Memo No: 5/13/87/2013/NCD-III) for providing essential fund in carrying out this project. We acknowledge Mr Tanmay Dolui, Indian Institute of Chemical Biology for Technical assistance in running flow cytometer.

Clinical trial identification

Legal entity responsible for the study

Indian Council of Medical Research.

Funding

Indian Council of Medical Research.

Disclosure

All authors have declared no conflicts of interest.