Understanding the Role of Macrophage-Tumor Cell Interaction in Breast Cancer Progression
Hanna, Samer J.
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While monocyte/macrophages are essential components of innate immunity, they can also have deleterious effects to the host in pathological conditions such as chronic inflammatory diseases or cancer. Tumor-associated macrophages (TAMS) are recruited in large numbers to many tumors where they critically interact with tumor cells and influence tumor formation, progression and resistance to chemotherapy. Therefore, it is highly important to comprehensibly characterize the mechanisms of macrophages recruitment and cellular interactions within the tumor microenvironment. Previous studies done by our group and others have shown that TAMS play a critical role in promoting tumor progression and invasion. Blocking this interaction and/or altering key macrophage signaling molecules reduces macrophage-dependent tumor cell invasion and metastasis both in vitro and in vivo. Prior to the interaction with tumor cells, macrophage recruitment from the circulation to the primary tumor is a multistep process known as diapedesis beginning with cell adhesion to the vascular endothelium, migration across the endothelial barrier (transendothelial migration), followed by degradation and migration through the sub-endothelial matrix. Macrophages among many types of immune cells form F-actin rich structures known as podosomes that are involved in cell adhesion, motility and degradation. Therefore, we were interested in understanding the mechanism by which macrophages are recruited from the circulation to the primary tumor and specifically in the distinct roles of podosomes during diapedesis. We investigated the roles of key regulators of actin cytoskeleton and podosome formation and function in response to important factors secreted by tumor cells namely Colony Stimulating Factor-1 (CSF-1) and IL-4. Factors that regulate podosome formation, the RhoGTPase Cdc42 and its downstream effector WASP a hematopoietic-specific protein, were important for the initial steps during diapedesis. However, podosome degradative function regulated by another downstream effector of Cdc42, N-WASP, as well as MT1-MMP metalloprotease was required for later steps following transendothelial migration. To further understand the role of Cdc42 in this process we developed a new FRET-based biosensor where we observed an initial surge in Cdc42 activation during podosome formation in macrophages. Using an in vitro tumor spheroid model, we then examined the role of podosome regulators in invasion towards tumor cells. We found that WASP and its phosphorylation by tyrosine kinase Hck are important for macrophage recruitment and infiltration into the tumor. Once the macrophages completed the process of diapedesis, they migrate directionally towards the tumor in a process known as chemotaxis. This is primarily driven by the secretion of CSF1 from the primary tumor which in turn induces macrophages to secrete Epidermal Growth Factor (EGF) that will stimulate tumor cells. In fact, blocking this paracrine interaction between the two cell types reduces tumor progression and metastasis in vivo. While the knowledge of cellular communication via these secreted factors has cast light on distant tumor–macrophage interactions, direct intercellular contact with TAMs within the complex and dense heterogeneous tumor matrix is still greatly underappreciated. Therefore, we investigated the direct cell-cell communication between macrophages and tumor cells and its effects on tumor cell functions. Recently, a novel mechanism of intercellular communication through long thin membranous tunneling nanotubes (TNTs) has been identified in many cell types including macrophages. TNTs can mediate the transfer of different materials including signaling molecules, nucleic acids, vesicles and organelles. In contrast to soluble factors that diffuse over distance, TNT propagate signals through a network of cells that remain robust despite the distance traveled. Moreover, TNTs have been suggested to play an important role in tumor microenvironments. However, very little is known about the formation, regulation and function of TNTs. Using advanced microscopy techniques such as live cell imaging, FRET-based biosensor technology and super-resolution microscopy we characterized the roles of multiple actin nucleating proteins in TNT formation and function including a key TNT-regulating protein called M-Sec, the Rho family GTPases Cdc42 and Rac1, along with downstream effectors WASP, N-WASP, WAVE2 and Arp2/3 complex. We next aimed understand the mechanism of tumor cell response to TNT-mediated interaction with macrophages. Using multiple in vitro assays that mimic the macrophage-tumor interaction in vivo, we found that macrophage TNTs mediated tumor cell elongation, co-invasion and cell streaming towards the endothelium. We then confirmed this in vivo using a zebrafish model and identified that macrophage TNTs were required for tumor cell invasion and dissemination. Overall, given the importance of tumor-stromai interactions, we believe that cur studies focused on macrophage recruitment and direct cell-cell interaction with tumor cells provide new insights that may lead to the development of pharmacoogical strategies for cancer treatment through the targeting of TNTs which may lead to more selective and effective therapies.