The interaction between tumor cells and macrophages is crucial in promoting tumor invasion and metastasis. In this study we examined a novel mechanism of intercellular communication, namely membranous actin-based tunneling nanotubes (TNTs) that occur between macrophages and tumor cells in the promotion of macrophage-dependent tumor cell invasion. The presence of heterotypic TNTs between macrophages and tumor cells induced invasive tumor cell morphology, which was dependent on EGF-EGFR signaling. Furthermore, reduction of a protein involved in TNT formation, M-Sec (TNFAIP2), in macrophages inhibited tumor cell elongation, blocked the ability of tumor cells to invade in 3D and reduced macrophage-dependent long distance tumor cell streaming in vitro. Using an in vivo zebrafish model that recreates macrophage mediated tumor cell invasion, we observed TNT mediated macrophage-dependent tumor cell invasion, distant metastatic foci and areas of metastatic spread. Overall, our studies support a role for TNTs as a novel means of interaction between tumor cells and macrophages that leads to tumor progression and metastasis.

The nonreceptor tyrosine kinase Pyk2 is highly expressed in invasive breast cancer, but the mechanism by which it potentiates tumor cell invasiveness is unclear at present. Using high-throughput protein array screening and bioinformatic analysis, we identified cortactin as a novel substrate and interactor of proline-rich tyrosine kinase 2 (Pyk2). Pyk2 colocalizes with cortactin to invadopodia of invasive breast cancer cells, where it mediates epidermal growth factor–induced cortactin tyrosine phosphorylation both directly and indirectly via Src-mediated Abl-related gene (Arg) activation, leading to actin polymerization in invadopodia, extracellular matrix degradation, and tumor cell invasion. Both Pyk2 and the closely related focal adhesion kinase (FAK) regulate tumor cell invasion, albeit via distinct mechanisms. Although Pyk2 regulates tumor cell invasion by controlling invadopodium-mediated functions, FAK controls invasiveness of tumor cells by regulating focal adhesion–mediated motility. Collectively, our findings identify Pyk2 as a unique mediator of invadopodium formation and function and also provide a novel insight into the mechanisms by which Pyk2 mediates tumor cell invasion.

Oncotarget | https://doi.org/10.18632/oncotarget.25243 Tomer Meirson, Alessandro Genna, Nikola Lukic, Tetiana Makhnii, Joel Alter, Ved P. Sharma, Yarong Wang, Abraham O. Samson, John S. Condeelis, Hava Gil-Henn Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors

Hepatocellular carcinoma (HCC) represents the fifth most common cancer worldwide and the third cause of cancer-related mortality. Hepatitis C virus (HCV) is the leading cause of chronic hepatitis, which often results in liver fibrosis, cirrhosis, and eventually HCC. HCV is the most common risk factor for HCC in western countries and leads to a more aggressive and invasive disease with poorer patient survival rates. However, the mechanism by which the virus induces the metastatic spread of HCC tumor cells through the regulation of invadopodia, the key features of invasive cancer, is still unknown. Here, the integration of transcriptome with functional kinome screen revealed that HCV infection induced invasion and invadopodia-related gene expression combined with activation of host cell tyrosine kinases, leading to invadopodia formation and maturation and consequent cell invasiveness in vitro and in vivo. The promotion of invadopodia following HCV infection was mediated by the sustained stimulation of epidermal growth factor receptor (EGFR) via the viral NS3/4A protease that inactivates the T-cell protein tyrosine phosphatase (TC-PTP), which inhibits EGFR signaling. Characterization of an invadopodia-associated gene signature in HCV-mediated HCC tumors correlated with the invasiveness of HCC and poor patient prognosis. These findings might lead to new prognostic and therapeutic strategies for virus-mediated invasive cancer.

Macrophages are important players involved in the progression of breast cancer, including in seeding the metastatic niche. However, the mechanism by which macrophages in the lung parenchyma interact with tumor cells in the vasculature to promote tumor cell extravasation at metastatic sites is not clear. To mimic macrophage-driven tumor cell extravasation, we used an in vitro assay (eTEM) in which an endothelial monolayer and a matrigel-coated filter separated tumor cells and macrophages from each other. The presence of macrophages promoted tumor cell extravasation, while macrophage conditioned media was insufficient to stimulate tumor cell extravasation in vitro. This finding is consistent with a requirement for direct contact between macrophages and tumor cells. We observed the presence of Thin Membranous Connections (TMCs) resembling similar structures formed between macrophages and tumor cells called tunneling nanotubes, which we previously demonstrated to be important in tumor cell invasion in vitro and in vivo. To determine if TMCs are important for tumor cell extravasation, we used macrophages with reduced levels of endogenous M-Sec (TNFAIP2), which causes a defect in tunneling nanotube formation. As predicted, these macrophages showed reduced macrophage-tumor cell TMCs. In both, human and murine breast cancer cell lines, there was also a concomitant reduction in tumor cell extravasation in vitro when co-cultured with M-Sec deficient macrophages compared to control macrophages. We also detected TMCs formed between macrophages and tumor cells through the endothelial layer in the eTEM assay. Furthermore, tumor cells were more frequently found in pores under the endothelium that contain macrophage protrusions. To determine the role of macrophage-tumor cell TMCs in vivo, we generated an M-Sec deficient mouse. Using an in vivo model of experimental metastasis, we detected a significant reduction in the number of metastatic lesions in M-Sec deficient mice compared to wild type mice. There was no difference in the size of the metastases, consistent with a defect specific to tumor cell extravasation and not metastatic outgrowth. Additionally, with an examination of time-lapse intravital-imaging (IVI) data sets of breast cancer cell extravasation in the lungs, we could detect the presence of TMCs between extravascular macrophages and vascular tumor cells. Overall, our data indicate that macrophage TMCs play an important role in promoting the extravasation of circulating tumor cells in the lungs.

Background: Metastasis is the cause of most cancer‐related deaths. It is known that breast cancer cells in proximity to macrophages become more invasive in an Epidermal Growth Factor (EGF) dependent manner. Tunneling nanotubes (TNTs) are thin, F–actin containing, cellular protrusions that mediate intercellular communication and have been identified in many tumors. The mechanism of TNT formation varies between different cell types. M‐Sec (TNFAIP2) has been demonstrated to be involved in TNT formation in some cell types including macrophages. Yet, the requirement of M‐Sec in tumor cell TNT formation in response to macrophages has not been explored.Aim: The aim of this study was to determine whether EGF was required for macrophage induced tumor cell TNTs in an M‐Sec dependent manner and what possible roles tumor cell TNTs play in tumor cell migration and invasion.Methods and Results: Macrophage Conditioned Media (CM) was used to induce an increase in TNTs in a number of breast cancer cell lines as measured by live cell microscopy. Tumor cell TNT formation by CM was dependent on the presence of EGF which was sufficient to induce TNT formation. CM treatment enhanced the level of M‐Sec identified using western blot analysis. Reduction of endogenous M‐Sec levels via shRNA in MTLn3 mammary adenocarcinoma cells inhibited the formation of TNTs. The role of tumor cell TNTs in cell behavior was tested using in vitro transwell and 3D invasion assays. No effect on chemotaxis was detected but 3D invasion was reduced following the knockdown of M‐Sec in tumor cell TNTs.Conclusions: Our results show that EGF was necessary and sufficient for tumor cell TNT formation which was dependent on cellular M‐Sec levels. While tumor cell TNTs may not play a role in individual cell behaviors like chemotaxis, they may be important in more complex tumor cell behaviors such as 3D invasion.