The role of the hypoxic microenvironment on the macrophagetumor cell interplay

Hypoxia is a common feature of solid tumors. Solid tumors microenvironment is composed by cancer cells, extracellular matrix (ECM), and stromal and immune cells that cooperate and affect each other. One of the most abundant cells at the tumor microenvironment are macrophages that can act as tumor suppressors (M1-like) or promotors (M2-like), depending on the stimuli they are exposed to. In order to understand how hypoxic tumor microenvironment affects the interplay between macrophages and tumor cells we established a co-culture system with a CRC cell line (RKO) and human macrophages, in normoxia (20%O2) and hypoxia (1%O2). Macrophages are monocyte-derived, isolated from healthy blood donors, and were cultured alone or with RKO cancer cells, under the conditions previously described. Additionally, RKO cancer cells was also cultured alone, under the same conditions. After co-culture, both populations were characterized by several techniques. First, mRNA expression of anhydrase carbonic IX (CAIX) was evaluated in RKO cancer cells to confirm that cells respond to hypoxia. Following, macrophage functionality and inflammatory profile were characterized by through several techniques. Concerning cancer cells, invasion-associated cellular activities were also evaluated. Macrophage characterization revealed that either cancer cells or hypoxia, individually or together, induced a mixed phenotype in macrophages, with both M1 and M2-like characteristics. Hypoxia by itself induce alterations in the expression of polarization markers, in the phagocytic activity and the expression of metabolism-related genes. However, the presence of cancer cells, is powerful than hypoxia in the modulation of macrophage cell area, metabolic activity and lactate/glucose metabolism. Nevertheless the combination of hypoxia and co-culture is able to potentiate the phosphorylation of NF-kB and IkBproteins. Concerning the effect of hypoxia and macrophages on cancer cells, we found that several mechanisms are involved in cancer cell response to these stimuli. We found in the conditions analyzed that hypoxia have a more powerful effect than the co-culture regarding the expression of metabolism-related genes and of some EMT-associated genes, as well as in Src and ERK proteins phosphorylation. We also observed that, in contrast, the co-culture with macrophages is powerful than hypoxia in modulating CD47 expression, lactate/ glucose metabolism and the expression of others EMT-associated genes. However the combination of hypoxia and co-culture with macrophages have a potentiated effect on CD47 expression, invasion and migration capacity of cancer cells and phosphorylation of Src and ERK proteins. FCUP The role of the hypoxic tumor microenvironment on the macrophage-tumor cell interplay 8 The model used in this work, an in vitro indirect co-culture system is a simple model to investigate the interaction between macrophages and cancer cells, under normoxic or hypoxic environment. It is an important tool to understand how cancer cells and macrophages connect to each other and how they respond to hypoxia, allowing to characterize the mechanisms involved in this response. Overall, we consider that the knowledge obtained with this work provide important information that might contribute to more efficient therapeutic approaches, either in re-educating tumor-associated macrophages, or targeting cancer cells, avoiding their invasion.