INEB THESIS DEFENSE: Novel therapeutic targets against cancer invasion: dissecting molecular mechanisms between macrophages and gastric cancer cells

INEB PhD student Patrícia Cardoso defended her thesis on May 5th, 2015 at FEUP, Faculty of Engineering of the University of Porto. The thesis ios untitled "Novel therapeutic targets against cancer invasion: dissecting molecular mechanisms between macrophages and gastric cancer cells" and was a work in the area of Bioengineering.
SUPERVISOR: Maria José Cardoso Oliveira, Assistant Investigator at INEB | CO-SUPERVISOR: Mário Adolfo Monteiro da Rocha Barbosa, Professor and Group Leader of the Microenvironments for NEWTherapies Group at INEB

Tumours are considered malignant when cancer cells fail to respect boundaries and invade the surrounding tissues. Invasive cancer cells may then, through lymph and blood vessels, reach secondary organs and metastasize. During this process, several interactions supporting invasion-related activities are established between cancer cells and stromal components of the tumour microenvironment. Macrophages, in particular, constitute a large portion of the tumour mass and are frequently associated with cancer progression. In breast cancer, macrophages are found at areas of basement membrane degradation or at the invasive front of advanced tumours.  A paracrine loop involving the production of colony-stimulating factor-1 (CSF-1) by breast cancer cells and of epidermal growth factor (EGF) by macrophages has been reported to modulate extracellular matrix (ECM) remodelling and intravasation of cancer cells into neighbour blood vessels. The role of macrophages in gastric and colorectal cancer progression, however, is still undetermined. Given that cancer cell invasion and macrophages are appealing and promising targets for anticancer therapies, this project aimed at i) identifying the major signalling pathways involved in the crosstalk between gastric and colon cancer cells and macrophages, ii) understanding how these molecular mechanisms contribute to cancer cell invasion and, finally, iii) designing a therapeutic strategy to target and impair these interactions.

This study demonstrated that soluble factors produced by human macrophages result in increased cancer cell invasion, motility and proteolytic activity and clarified the underlying molecular mechanisms. EGF was identified as a key pro-invasive and pro-motile factor produced by macrophages, with the ability to activate cancer cell EGFR signalling pathway, leading to increased motility and invasion. This signalling pathway involved the phosphorylation of Akt, c-Src and ERK1/2, and led to an increase of RhoA and Cdc42 smallGTPase activity. Interestingly, whereas macrophage-mediated cancer cell c-Src and ERK1/2 phosphorylation occurred downstream EGFR phosphorylation/activation, Akt phosphorylation seems to be a parallel event, taking place in an EGFR-independent manner.

Moreover, this study evidenced that the role of macrophages on gastric and colon cancer cell-related activities was dependent on macrophage phenotype. The anti-inflammatory M2-like macrophages were more efficient in stimulating cancer cell invasion, motility/migration and angiogenesis than their pro-inflammatory M1-like counterparts. Interestingly, both macrophage populations similarly induced EGFR tyrosine phosphorylation and activation of its downstream partners. Distinct abilities in promoting invasion-related activities were due to differences in matrix metalloproteinase activity.

These results inspired the design of a therapeutic strategy aiming to counteract macrophage pro-tumour activities and, simultaneously, to reverse their differentiation from an M2-like anti-inflammatory into an M1-like pro-inflammatory phenotype. Therefore, interferon-γ, a pro-inflammatory cytokine known to modulate macrophage differentiation, was successfully incorporated into chitosan/poly(γ-glutamic acid)-based delivery systems. The gradual cytokine release was effective in modulating macrophage morphology, cytoskeleton organization and cytokine profile. Most interestingly, this strategy successfully reversed the stimulation of invasion provided by M2-like macrophages.

In summary, this work highlighted the role of the tumour microenvironment, and in particular of macrophages, in gastric and colon cancer cell-invasion, motility, proteolysis and angiogenesis, identifying, for the first time, the underlying molecular mechanisms. The contribution for such cellular activities of macrophage populations with distinct inflammatory profiles was also elucidated. Additionally, a strategy to modulate macrophage pro-invasive activity at the tumour microenvironment was proposed, as groundwork for a therapeutic approach targeting cancer progression