Novel immunotherapeutic mechanisms to limit lung metastases in breast cancer

Current therapies for breast cancer depend primarily on chemicals that kill cells (hence the term chemotherapy). These chemicals are not specific to cancer cells; they are also toxic to normal cells. This means that they have unpleasant and sometimes dangerous side effects, especially on the intestine and on the immune system. These toxic side effects limit the amount of cancer killing chemicals that can be used in patients.To get around this problem, some researchers are looking for chemicals that will only kill cancer cells. This has proven very difficult to do. Our approach is to stimulate the immune system to fight the cancer. This is called “immunotherapy” and takes advantage of the body’s own immune defence mechanisms to inhibit cancer growth. In this way the use of toxic chemicals can be reduced. Using our extensive knowledge of the immune system (the principal investigator has been working in this field for 30 years) we have developed an experimental plan that uses an extract of the fungus Cordyceps sinensis to activate a component of the immune system. Our work to date has shown that this extract, given orally, can reduce breast cancer metastases by up to 80%. The experiments outlined in this proposal will provide information on the agent in the extract responsible for this effect and the mechanism by which it occurs. Isolate the active agent in the immunotherapeutic extractInitially we will confirm or refute our hypothesis that the active agent is a polysaccharide. We will first obtain a crude polysaccharide extract (CPE) from our aqueous extract. This initial CPE will be tested at various concentrations for its ability to activate MØ to elaborate factors that limit the growth of breast cancer cells in vitro. If CPE proves successful we will begin the process of isolating and characterizing the active moiety. We will first use NMR spectroscopy to give us information on the purity of our polysaccharide extract and reveal peptide content. If glycoproteins are found, we will undertake proteolytic cleavage using enzymes immobilized on beaded agarose. The digested material will be harvested for our in vitro assay. Subsequent to the NMR analysis we will subject the polysaccharide extract to a preliminary size exclusion analysis. Following this size exclusion step, we will use charge separation chromatography using DEAE cellulose. The second, and more sophisticated, technique will be to use hydrophilic interaction liquid chromatography (HILIC).Identification of the factors responsible for the MØ-mediated anti-neoplastic effectTo identify the soluble effector factors released by the activated MØ we will first screen the activated MØ using microarray analysis to reveal genes that are highly upregulated by stimulation by the Cs extract (and components). The data acquired from microarray will be confirmed using quantitative RT-PCR (qPCR), as we have previously described, where we will focus on a smaller number of relevant genes (approximately 10). The expression data will be confirmed using ELISA analysis of the supernatants of stimulated MØ to confirm the presence of secreted protein. Once we establish the array of factors that are being produced by the stimulated MØ, we will investigate which of these factors are having an anti-neoplastic effect, using siRNA knock-down and add-back strategies for likely target proteins in the culture supernatants. To confirm the importance of the factors identified, we will use knock-out animals (where possible) in our in vivo breast cancer model.