4-year PhD Studentship: Targeting cancer stem cells: Selective culture method and drug search

Cancer stem cells are a small population of cells in tumours maintaining the tumorigenic capacity (1). They are featured as quiescent (i.e., dormant), in contrast to the majority of cancer cells that actively proliferate under the aberrant regulation. Most cancer therapies target fast-proliferating cells to suppress tumour growth. Due to the quiescent feature, cancer stem cells are resistant to most of the current chemo- and radiotherapies, thus serving as the main cause of relapse. Cancer stem cells are also thought to be the most effective form of making new tumours in metastasis. As such, the development of anti-cancer drugs that specifically target cancer stem cells is desired to prevent cancer recurrence.

In collaboration with Nissan Chemical Corporation, we developed an in vitro culture supplement which facilitates clonal 3D spheroidal cultures allowing a large-scale chemical screening using liquid-handling robots (2). We also characterised such-grown cancer spheroids (3) and found that the culture method can be used for the growth of cancer stem cells (unpublished).

We currently examine the molecular mechanism for the induction and maintenance of stemness. We wish to apply the obtained knowledge to the actual drug screening.

Aims and Objectives

Aim: To find chemical drugs that specifically block the growth and/or maintenance of cancer stem cells

Objectives:

• Refine the culture condition in which cancer stem cells are most effectively induced and maintained in the 3D culture, which would allow us to conduct drug screening

The finding will also be beneficial in the study of the microenvironment required for cancer stem cells.

• Once candidate chemical molecules are selected, validate the effect as cancer stem cell-targeting drugs
• On the validated molecules, elucidate the mechanism of action whereby they block the growth and/or maintenance of cancer stem cells

Methodology

Well-characterised breast cancer cells MCF7 will be cultured as described previously (2, 3). A refined culture condition that induces and enriches stemness will be searched by testing various lengths of cultures and ingredients. Stemness will be assessed by the expression of genes including Nanog, Oct4, CD24 and CD44 using qPCR, immunocytochemistry and fluorescence-activated-cell-sorting (FACS). Our preliminary data suggest that culturing 7 days without serum or less than 4 days with serum shows up-regulation of Nanog and Oct4. Further refinement will be required.

Once the culture method is optimised, the chemical screening will be conducted by Nissan using a library consisting of chemicals with biological and pharmacological activities, with their wealth of expertise in drug screening.

Isolated chemicals will be validated by gene expression and cell proliferation. Anticipated mechanisms include (i) killing stem cells by targeting stemness-specific signal transduction and (ii) suppressing stemness by inducing cell proliferation and differentiation. Given that stem cells are mostly at the G0/G1 phase of the cell cycle, apoptosis will be examined for case (i). For (ii), differentiation markers (CK8, CK18) and de-differentiated markers (CK5, CK17) will be examined along with the loss of stem markers. The sensitivity to conventional anti-cancer drugs will also be examined.

Keywords

Cancer stem cells, drug screening, in vitro cell culture

How to apply for this project

This project will be based in Bristol Veterinary School in the Faculty of Health Sciences at the University of Bristol.

Please visit the Faculty of Health Sciences website for details of how to apply