Invasion of tumor cells into the surrounding connective tissue and blood vessels is a key step in the metastatic spread of breast tumors. Although the presence of macrophages in primary tumors is associated with increased metastatic potential, the mechanistic basis for this observation is unknown. Using a chemotaxis-based in vivo invasion assay and multiphoton-based intravital imaging, we show that the interaction between macrophages and tumor cells facilitates the migration of carcinoma cells in the primary tumor. Gradients of either epidermal growth factor (EGF) or colony-stimulating factor 1 (CSF-1) stimulate collection into microneedles of tumor cells and macrophages even though tumor cells express only EGF receptor and macrophages express only CSF-1 receptor. Intravital imaging shows that macrophages and tumor cells migrate toward microneedles containing either EGF or CSF-1. Inhibition of either CSF-1-or EGF-stimulated signaling reduces the migration of both cell types. This work provides the first direct evidence for a synergistic interaction between macrophages and tumor cells during cell migration in vivo and indicates a mechanism for how macrophages may contribute to metastasis.

Although the presence of macrophages in tumors has been correlated with poor prognosis, until now there was no direct observation of how macrophages are involved in hematogenous metastasis. In this study, we use multiphoton microscopy to show, for the first time, that tumor cell intravasation occurs in association with perivascular macrophages in mammary tumors. Furthermore, we show that perivascular macrophages of the mammary tumor are associated with tumor cell intravasation in the absence of local angiogenesis. These results show that the interaction between macrophages and tumor cells lying in close proximity defines a microenvironment that is directly involved in the intravasation of cancer cells in mammary tumors. [Cancer Res 2007;67(6):2649-56]

Most invasive solid tumours display predominantly collective invasion, in which groups of cells invade the peritumoral stroma while maintaining cell-cell contacts. As the concepts and experimental models for functional analysis of collective cancer cell invasion are rapidly developing, we propose a framework for addressing potential mechanisms, experimental strategies and technical challenges to study this process.

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Previous studies have shown that macrophages and tumor cells are comigratory in mammary tumors and that these cell types are mutually dependent for invasion. Here we show that macrophages and tumor cells are necessary and sufficient for comigration and invasion into collagen I and that this process involves a paracrine loop. Macrophages express epidermal growth factor (EGF), which promotes the formation of elongated protrusions and cell invasion by carcinoma cells.

Responses of tethered cells ofEscherichia coil to impulse, step, exponential-ramp or exponentiated sine-wave stimuli are internally consistent, provided that allowance is made for the nonlinear effect of thresholds. This result confirms that wild-type cells exposed to stimuli in the physiological range make short-term temporal comparisons extending 4 sec into the past: the past second is given a positive weighting, the previous 3 sec are given a negative weighting, and the cells respond to the difference. cheRcheB mutants (defective in methylation and demethylation) weight the past second in a manner similar to the wild type, but they do not make short-term temporal comparisons. When exposed to small steps delivered iontophoretically, they fail to adapt over periods ofup to 12 sec; when exposed to longer steps in a flow cell, they partially adapt, but with a decay time of >30 sec. cheZ mutants use a weighting that extends at least 40 sec into the past. The gain of the chemotactic system is large: the change in occupancy of one receptor molecule produces a significant response.Flagellated bacteria, such as Escherichia coli, possess a stimulus-response system in which the input is the local concentration of a chemical and the output is the motion of the cell. Some chemicals, such as aspartate, are sensed by specific receptors (1). Changes in their occupancy lead to changes in the probability that the flagella spin clockwise (CW) or counterclockwise (CCW). This determines whether the cell moves erratically with little net displacement (tumbles) or swims smoothly (runs), respectively (2, 3). These modes alternate, causing the cell to move about as in a random walk (4). When the occupancy of the aspartate receptor increases, the probability of CCW rotation increases, the flagella tend to remain in a bundle that pushes the cell forward, and the cell continues to run. Thus, in a spatial gradient of attractant, the random walk is biased: runs that happen to carry the cell up the gradient are extended. This enables the cell to move to a more favorable environment.The A variety of methods have been used to monitor the chemotactic behavior of wild-type and mutant bacterial strains. The simplest involves observation of chemotactic rings on agar plates; however, ring formation requires transport, metabolism, and growth, in addition to chemotaxis (6). Another method involves the addition of a large amount of an attractant or a repellent to a population of swimming (or tethered) cells. These respond by swimming smoothly or tumbling (or spinning CCW or CW) for a relatively long period of time and then they eventually recover (2, 7). In wild-type cells, the time required for recovery is proportional to the net change in receptor occupancy (8,9). A third method follows the accumulation of cells in capillary tubes containing attractants at different concentrations (10). Some other methods are less convenient but provide additional information. These include measurements of drift rates of cells in layered gradients (11, 12)...

We subjected cells collected using an in vivo invasion assay to cDNA microarray analysis to identify the gene expression profile of invasive carcinoma cells in primary mammary tumors. Expression of genes involved in cell division, survival, and cell motility were most dramatically changed in invasive cells indicating a population that is neither dividing nor apoptotic but intensely motile. In particular, the genes coding for the minimum motility machine that regulates ␤-actin polymerization at the leading edge and, therefore, the motility and chemotaxis of carcinoma cells, were dramatically up-regulated. However, ZBP1, which restricts the localization of ␤-actin, the substrate for the minimum motility machine, was down-regulated. This pattern of expression implicated ZBP1 as a suppressor of invasion. Reexpression of ZBP1 in metastatic cells with otherwise low levels of ZBP1 reestablished normal patterns of ␤-actin mRNA targeting and suppressed chemotaxis and invasion in primary tumors. ZBP1 reexpression also inhibited metastasis from tumors. These experiments support the involvement in metastasis of the pathways identified in invasive cells, which are regulated by ZBP1.

Invadopodia are actin-rich membrane protrusions with a matrix degradation activity formed by invasive cancer cells. We have studied the molecular mechanisms of invadopodium formation in metastatic carcinoma cells. Epidermal growth factor (EGF) receptor kinase inhibitors blocked invadopodium formation in the presence of serum, and EGF stimulation of serum-starved cells induced invadopodium formation. RNA interference and dominant-negative mutant expression analyses revealed that neural WASP (N-WASP), Arp2/3 complex, and their upstream regulators, Nck1, Cdc42, and WIP, are necessary for invadopodium formation. Time-lapse analysis revealed that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours. Invadopodia with short lifetimes are motile, whereas long-lived invadopodia tend to be stationary. Interestingly, suppression of cofilin expression by RNA interference inhibited the formation of long-lived invadopodia, resulting in formation of only short-lived invadopodia with less matrix degradation activity. These results indicate that EGF receptor signaling regulates invadopodium formation through the N-WASP–Arp2/3 pathway and cofilin is necessary for the stabilization and maturation of invadopodia.