Macrophages Promote Tumor Cell Extravasation across an Endothelial Barrier through Thin Membranous Connections

Abstract

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 (Hanna 2019). 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, examination of time-lapse intravital-imaging (IVI) data sets of breast cancer cell extravasation in the lung, 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 lung.

CD13 Activation Assembles Signaling Complexes that Promote the Formation of Tunneling Nanotubes in Endothelial Cells

“…Interestingly, TNTs have been implicated in both inflammation (Jiang et al ., 2016; Weng et al ., 2016; Ariazi et al ., 2017; Nawaz and Fatima, 2017; Weidle et al ., 2018; Yao et al ., 2018; Kuret et al ., 2019; Mittal et al ., 2019a) and angiogenesis (Figeac et al ., 2014; Climent et al ., 2015; Patheja and Sahu, 2017; Errede et al ., 2018; Lou et al ., 2018; Liu et al ., 2019), indicating that CD13 may be upregulated specifically to participate in these processes, possibly by promoting TNT formation and function. We have also determined that during inflammation, activation of CD13 on endothelial cells increases monocyte adhesion and transmigration (Mina-Osorio et al ., 2008; Subramani et al ., 2013; Ghosh et al ., 2014; Rahman et al ., 2014a; Rahman et al ., 2014b), which may relate to “transmigratory cups” or “endothelial adhesive platforms” which are actin-based, TNT-like docking protrusions that form at the site of inflammatory leukocyte extravasation to mediate the adhesion phase of immune infiltration (Carman et al ., 2003; Carman and Springer, 2004; Riethmuller et al ., 2008; Wittchen, 2009; Vestweber et al ., 2013; Franz et al ., 2016; Johnson et al ., 2017; Genna et al ., 2023). In support of this notion, we have found that endothelial CD13 is highly enriched in similar protrusions formed between monocytes and endothelial cells in vitro (Mina-Osorio et al ., 2008).…”

“…Interestingly, overexpression of NG2 in cancer cell lines stimulates TNT formation (Lou et al ., 2018), consistent with inducible angiogenic regulators as potential mediators of TNT function. It will be interesting to investigate CD13 dependent TNT formation in heterotypic co-cultures of myeloid and endothelial cells or tumor cells (Genna et al ., 2023).…”

“…Indeed, the microenvironment of damaged tissues is highly toxic and proteolytic, yet transplanted mesenchymal stem cells are able to transfer healthy organelles and vesicles to rescue damaged cells, suggesting a more ‘protected’ means of communication must exist. Alternatively, protrusion-based cellular interactions such as filopodia (Gardel et al ., 2010; Jacquemet et al ., 2015), cytonemes (Kornberg and Roy, 2014; Stanganello and Scholpp, 2016) and although less well studied, the tunneling nanotubes (TNTs) (Onfelt et al ., 2004; Rustom et al ., 2004; Baker, 2017; Yamashita et al ., 2018; Hanna et al ., 2019; Genna et al ., 2023) would provide specificity over soluble- or exosome-based intercellular communication mechanisms (Caviglia and Ober, 2018; Yamashita et al ., 2018). Filopodia are short, thin, actin-containing protrusions localized at the cell base with important roles in sensory and cell-cell communication (Jacinto and Wolpert, 2001; Fischer et al ., 2019), while cytonemes are long, close-ended specialized filopodia that connect distant cells to transport signals between various cell types in Drosophila and zebrafish (Gonzalez-Mendez et al ., 2019), (Eom et al ., 2015), (Hamada et al ., 2014), (Mattes et al ., 2018).…”

Ex Vivo Models to Study the Interaction of Immune Cells and Cancer Cells under Therapy

“…Intravital imaging of cancer has provided essential insight into the diverse steps of metastasis such as intravasation (19,20), extravasation (21)(22)(23) and seeding (21) and outgrowth (24) of metastatic cells. These studies have shown that under chronic lung in ammation, neutrophils release extracellular traps that can modulate the microenvironment to awaken the dormant cancer cells (24); that macrophages can facilitate the dissemination of cancer cells by modulating vascular opening at TMEM doorways, allowing motile cancer cells to spread hematogenously (19); and that metastatic dissemination can sometimes be enhanced by neoadjuvant chemotherapy (25).…”