Cancer Metabolism

Project 1: Examine the role of immunosuppressive metabolites in lymphocyte viability

Introduction

Brain cancer is the leading cause of cancer death in people under 15 and 39 years of age.  Brain cancer cells evolve to become immunosuppressive, which is central to their survival and growth. One mechanism of this immune suppression is activation of a metabolic pathway, kynurenine pathway (KP).  This activation plays a pivotal role in suppressing anti-tumour immunity and supporting tumour growth resulting partly from the direct effects of accumulating KP-derived immunosuppressive metabolites.  However, the effect of the complete KP metabolic profile on lymphocyte viability remains to be investigated.

Hypothesis:

Brain cancer patients are notable for having profound immunosuppression and we hypothesise that:

  1. Brain cancer cells produce KP-derived metabolites.
  2. Brain cancer derived KP metabolites demonstrate immunosuppressive capacity in vitro.

Aims:

  1. Characterise the KP metabolic profile in brain cancer cells isolated from patient tissue.
  2. Evaluate the direct immunosuppressive effects of specific KP metabolites by:
  3. Culturing and phenotyping specific lymphocyte subsets
  4. Examining the direct effect of KP metabolites and brain cancer cell supernatant on lymphocyte proliferation and apoptosis.

Research Plan:

Brain cancer cells will be isolated from surgically resected patient tissue by methods previously published. Evaluation of KP metabolites in the brain cancer cell culture supernatants will be assessed using LC/MS/MS. To investigate the immunosuppressive properties of brain cancer cells, we will use two different experimental approaches. In the first approach, the supernatants from brain cancer cells will be used in culture assays with lymphocytes from matching patient blood. In the second approach, we will add KP metabolites directly into lymphocyte cultures.

Lymphocytes will be isolated from patient blood using methods previously described in literature. In approach one, conditioned medium obtained from 3-day cultures of brain cancer cells will be added to the lymphocytes and incubated for 5 days. In approach two, serial dilutions of KP metabolites and vehicle controls will be added to the lymphocytes and cultured for 3-7 days. For evaluation of cell proliferation, lymphocytes will be labelled with the CFSE dye and lymphocyte apoptosis will be assessed by the Annexin V/PI staining kit. Assessment of the level of immunosuppression exerted by the KP metabolites and brain cancer cell supernatant will be performed by flow cytometry. Phenotyping of lymphocyte populations will be performed by staining cells with a panel of intracellular and surface markers.  

Enquires:
Dr Seray Adams / Professor Gilles Guillemin
Email: seray.adams@mq.edu.au / gilles.guillemin@mq.edu.au