1. Are macrophages interesting targets for anti-cancer therapy?
Maria Oliveira
Lisbon, July 4th, 2010

2. Invasion: an ideal target for anti-cancer therapy
So, first a brief introduction about the meaning of invasion in the context of cancer, and why are we focusing on this subject.
In the case of solid cancers, invasion occurs when genetically altered cells cross their tissue limits, degrade the basement membrane, reach the underlying stroma, enter blood vessels and generate, at distant sites new focus of cancer, generally called metastasis.
These are responsible for the great majority of cancer-related deaths.
Invasion is therefore the hallmark of malignancy and one of the most appealing targets for anti-cancer therapy. If one could counteract invasion, cancer would not progress and metastasis would not occur.
Adapted from Robbins and Cotran, 7th Edition

3. The tumour microenvironment
So if different types of cells are present at the tumour microenvironment why did we choose to focus on macrophages?
Macrophages are considered obligate partners for cancer cell migration, invasion, and metastasis, playing an active role in this process (breast cancer and melanoma models).
Tumors direct macrophages to contribute to invasion, intravasation, angiogenesis, metastasis formation and matrix remodelling.

4. Macrophages: obligate partners for cancer cell invasion and metastasis

5. Macrophages: central targets for anti- cancer therapy
Macrophages: dual role in cancer
Macrophages: central targets for anti- cancer therapy
Tumor supression
Tumor promotion

6. Short-term objectives
• clarify the role of macrophages on gastric cancer cell invasion;
• identify the major signalling pathways activated during the molecular crosstalk between invading gastric cancer cells and surrounding macrophages;
• select candidate molecules suitable for therapeutical approach.

7. Long-term objective: anti-cancer therapy
Nanoparticles
Specific inhibitors
The general idea of our long term objective is to adsorb, at the surface of pre-designed nanoparticles, inhibitors of selected invasion-related signalling pathways.
Such immobilized inhibitors will then be uptake by the circulating monocytic population.
The high inflammatory response at the tumour primary site will call such monocytes to the tissue and induce their differentiation into macrophages
Monocyte / Macrophage

8. Long-term objective: anti-cancer therapy
Afterwards, the inhibitor-adsorbed nanoparticles will be released by activated macrophages, at the tumour site. Another strategy would be the modulation of the macrophage polarization towards a tumouricidal phenotype or to block the pro-tumoural polarization.
Before such approach, it is however important to understand the molecular crosstalk and signalling pathways in all it’s complexity established at the
primary site between cancer cells and macrophages. 8

9. M1 and M2 macrophage modulation
Cytokine profile
Morphology
Cell surface receptors

10. Altered differentiation program
Monocytes M2
Time
Monocytes
LPS
Altered differentiation program M1

11. Macrophages stimulate gastric cancer cell invasion into ECM components
Matrigel Invasion Assay

12. MMPs are involved in macrophage-mediated cancer cell invasion
LPS-treated macrophages (M1) are less efficient in stimulating cancer cell invasion probably due to their reduced proteolytic activity.

13. Macrophages stimulate gastric cancer cell motility / migration
AGS + RPMI
AGS + cmMac
AGS + cmMac
AGS + RPMI

14. Inhibition targeting MMPs and EGFR reduced macrophage-mediated invasion
CSF-1
Macrophage
Breast cancer cell

15. Macrophages enhance cancer cell EGFR tyrosine phosphorylation (Y1086)

16. EGFR – silencing related molecules (siRNA) on gastric cancer cells
Signalling pathways related to cell survival, cell motility, gene regulation, cytoskeleton regulation

17. PLC-, GAB1 and Cbl are required for macrophage-mediated invasion

18. Macrophage enhance cancer cell EGFR, PLC-, c-Src, Akt and Erk phosphorylation

19. Altered differentiation program
Monocytes M2
Time
Monocytes
LPS
Altered differentiation program M1
Epithelial cell M2
Signalling pathways
EGFR
MMP
ROCK
PI3K
PLC-gama
CBL GAB1
SHC
Grb2
FAK
Silencing
pEGFR
pAKT
pSrc
pPLC-gama
pERK =
Invasion
Migration

20. Acknowledgments INEB Mário Barbosa Ana Patrícia Cardoso
Marta Oliveira
Perpétua Pinto do Ó
Susana Santos
Catarina Almeida
Raquel Gonçalves
Outdoors Collaborators
Marc Mareel (UGhent, Belgium)
Alberto Mantovani (ICH, Milan, Italy)
Tobias Pukrop
Claudia Binder (Georg-August University,
Goettingen, Germany)
IPATIMUP
Raquel Seruca
Fátima Carneiro
IBMC
João Relvas
Ana Filipa Gonçalves