We report a technique to evaluate the same tumor microenvironment over multiple intravital imaging sessions in living mice. Individual tumor cells expressing photoswitchable proteins in an orthotopic mammary carcinoma are optically marked and followed for extended times through a Mammary Imaging Window (MIW). We demonstrate, for the first time, that two distinct microenvironments in the same orthotopic mammary tumor affect differently the invasion and intravasation of tumor cells. KeywordsTumor microenvironment; photoswitching; photoactivatable fluorescent proteins; migration; invasion; intravasation; intravital imaging; metastasis; mammary imaging window The early steps of metastasis are characterized by tumor cells invading the stroma (invasion) and entering the blood (intravasation)1 , 2. Short term tracking of individual cells inside fluorescent tumors by intravital imaging has revealed dramatic heterogeneity in tumor cell invasion and intravasation3. However, long term tracking of individual cells is required to quantify these behaviors and to determine the fates of cells in specific tumor microenvironments. Imaging techniques that rely on surgical dissection to expose the imaging site have limitations for long-term experiments such (1) tissue dehydration, impaired thermoregulatory control and/or animal survival upon surgical dissection, (2) possible effects of prolonged anesthesia exposure, and (3) a limited field of view. These limitations can be overcome by studying tumors through a dorsal skinfold chamber4. The use of dorsal skinfold chambers, however, limits the experiments to tumor-models based on cell lines, and for many tumors a non-orthotopic environment. For example, invasion and intravasation of breast tumor cells is highly dependent on the specific local microenvironment5 which may not exist in the Correspondence to: John Condeelis, condeeli@aecom.yu.edu; Jeffrey E. Segall, segall@aecom.yu.edu; Jacco van Rheenen, j.vanrheenen@niob.knaw.nl. 4 DK and BG contributed equally Author contributions: DK, BG, and JvR, JES, JC contributed equally to this manuscript, and they conceived of and designed the study, performed the imaging, and analysis and drafted the manuscript. JW advised and trained in intravital imaging, VVV provided materials and advice. To image orthotopic (in the natural, mammary gland environment) breast tumors intravitally at high resolution for prolonged times, we developed a MIW that can be placed on top of the mammary gland of a mouse (Fig. 1a). The protocol for these animal studies was approved by the Institutional Animal Care and Use Committee for the Albert Einstein College of Medicine. The MIW consists of two plastic rings which form a mount for a glass coverslip. The mount has holes which facilitate suturing into the skin, whereas the glass coverslip assures the optimal working distance and refraction index for high resolution imaging (for more details on the equipment used for imaging, see Supplementary Fig. 1 online). While surgical dissection of the skin overlaying t...

SummaryWe have shown previously that distinct Mena isoforms are expressed in invasive and migratory tumor cells in vivo and that the invasion isoform (Mena INV ) potentiates carcinoma cell metastasis in murine models of breast cancer. However, the specific step of metastatic progression affected by this isoform and the effects on metastasis of the Mena11a isoform, expressed in primary tumor cells, are largely unknown. Here, we provide evidence that elevated Mena INV increases coordinated streaming motility, and enhances transendothelial migration and intravasation of tumor cells. We demonstrate that promotion of these early stages of metastasis by Mena INV is dependent on a macrophage-tumor cell paracrine loop. Our studies also show that increased Mena11a expression correlates with decreased expression of colony-stimulating factor 1 and a dramatically decreased ability to participate in paracrine-mediated invasion and intravasation. Our results illustrate the importance of paracrine-mediated cell streaming and intravasation on tumor cell dissemination, and demonstrate that the relative abundance of Mena INV and Mena11a helps to regulate these key stages of metastatic progression in breast cancer cells. Journal of Cell Science metastatic progression. Mena is a member of the Ena/VASP family of proteins and binds actin to regulate the geometry and assembly of filament networks through: (1) an anti-capping protein activity (Bear et al., 2002;Barzik et al., 2005; Hansen and Mullins, 2010) that involves binding to profilin and both G-and F-actin; (2) Mena tetramerization, and (3) reduction in the density of actin-related proteins 2 and 3 (Arp2/3)-mediated branching (Gertler et al., 1996;Barzik et al., 2005;Ferron et al., 2007;Pasic et al., 2008;Bear and Gertler, 2009; Hansen and Mullins, 2010). Alternative splicing for the Mena gene has been reported: a 19 amino acid residue insertion just after the EVH1 domain generates the Mena invasion isoform (Mena INV , formerly Mena +++ ) (Gertler et al., 1996;Philippar et al., 2008), whereas a 21 residue insertion in the EVH2 domain generates the Mena11a isoform (Di Modugno et al., 2007). A comparison of the invasive and migratory tumor cells collected in vivo, with primary tumor cells isolated from mouse, rat and human cell-line-derived mammary tumors, revealed that Mena INV expression is upregulated and Mena11a is downregulated selectively in the invasive and migrating carcinoma cell population (Goswami et al., 2009). The differential regulation of Mena isoforms across species suggests that these two isoforms have important roles in invasion and metastasis.In previous studies, we showed that expression of Mena INV in a xenograft mouse mammary tumor promotes increased formation of spontaneous lung metastases from orthotopic tumors and alters the sensitivity of tumor cells to epidermal growth factor (EGF) . We undertook the current study to identify the step(s) in the metastatic cascade that are affected by Mena INV expression and investigate the effect of expression of...

SummaryInvadopodia are proteolytic membrane protrusions formed by highly invasive cancer cells, commonly observed on substrate(s) mimicking extracellular matrix. Although invadopodia are proposed to have roles in cancer invasion and metastasis, direct evidence has not been available. We previously reported that neural Wiskott-Aldrich syndrome protein (N-WASP), a member of WASP family proteins that regulate reorganization of the actin cytoskeleton, is an essential component of invadopodia. Here, we report that N-WASP-mediated invadopodium formation is essential in breast cancer invasion, intravasation and lung metastasis. We established stable cell lines based on MTLn3 rat mammary adenocarcinoma cells that either overexpressed a dominant-negative (DN) N-WASP construct or in which N-WASP expression was silenced by a pSuper N-WASP shRNA. Both the N-WASP shRNA and DN N-WASP cells showed a markedly decreased ability to form invadopodia and degrade extracellular matrix. In addition, formation of invadopodia in primary tumors and collagen I degradation were reduced in the areas of invasion (collagen-rich areas in the invasive edge of the tumor) and in the areas of intravasation (blood-vessel-rich areas). Our results suggest that tumor cells in vivo that have a decreased activity of N-WASP also have a reduced ability to form invadopodia, migrate, invade, intravasate and disseminate to lung compared with tumor cells with parental N-WASP levels.

We report on the development of a new SYBR Green I-based plate assay for analyzing the activities of antimalarial drugs against intraerythrocytic Plasmodium falciparum. This assay is considerably faster, less labor-intensive, and less expensive than conventional radiotracer (e.g., [3 H]hypoxanthine and [ 3 H]ethanolamine)-based assays or P. falciparum lactate dehydrogenase activity-based assays. The assay significantly improves the pace at which antimalarial drug discovery efforts may proceed.The continued emergence and spread of multidrug-resistant strains of Plasmodium falciparum and P. vivax are arguably the most pressing problems in the area of infectious diseases today. Also, although the recent deciphering of the P. falciparum genome reveals many promising new drug targets, the financial cost of bringing drugs to the clinic is a major obstacle in the development of new antimalarials (6). A faster, less expensive, high-throughput means of screening the activities of drugs against a variety of malarial parasite strains would greatly assist preclinical drug development.Quantitative assessment of the effects of drugs on parasite growth and development can be achieved by direct (but extremely tedious) microscopic examination of blood smears. An alternative assay is measurement of the effect of drug exposure by determination of the level of incorporation of radiolabeled hypoxanthine. While the latter method can be automated, it requires radioactive materials and is not convenient for detection of parasite stage-specific effects. Another assay measures parasite lactate dehydrogenase activity by methods that do not require radioisotopes. However, this assay requires multiple processing steps and expensive reagents and is not particularly cost-effective for large-scale drug screening efforts.We have thus endeavored to develop more rapid and convenient cell-based assays for quantifying antimalarial drug activities. We have strived to enhance simplicity and reduce cost. In this paper, we report on the development of one such assay that relies on the fluorophore SYBR Green I. MATERIALS AND METHODSCell culture. Asexual culture is routinely performed. Parasite cultures are initiated from stabilates preserved in liquid nitrogen (the level of parasitemia during storage is Ն10%). Following the initiation of a fresh culture, at least two full life cycles (96 h) are completed before parasites are used for assays. In general, cultures are synchronized in the laboratory, and assays are initiated when the parasites are at the ring stage. However, we find that this assay is equally applicable to asynchronous culture and that similar 50% inhibitory concentrations (IC 50 s) are calculated from data with asynchronous and synchronous cultures (data not shown). Prior to assay initiation, the level of parasitemia of an aliquot of a stock culture is measured by light microscopy following Giemsa staining or by fluorescence-activated cell sorter analysis after staining with propidium iodide. In general, stock cultures with 5 to 10% paras...