SummaryMost cancer cells release heterogeneous populations of extracellular vesicles (EVs) containing proteins, lipids, and nucleic acids. In vitro experiments showed that EV uptake can lead to transfer of functional mRNA and altered cellular behavior. However, similar in vivo experiments remain challenging because cells that take up EVs cannot be discriminated from non-EV-receiving cells. Here, we used the Cre-LoxP system to directly identify tumor cells that take up EVs in vivo. We show that EVs released by malignant tumor cells are taken up by less malignant tumor cells located within the same and within distant tumors and that these EVs carry mRNAs involved in migration and metastasis. By intravital imaging, we show that the less malignant tumor cells that take up EVs display enhanced migratory behavior and metastatic capacity. We postulate that tumor cells locally and systemically share molecules carried by EVs in vivo and that this affects cellular behavior.

SummaryForced overexpression and/or downregulation of proteins regulating epithelial-to-mesenchymal transition (EMT) has been reported to alter metastasis by changing migration and stem cell capacity of tumor cells. However, these manipulations artificially keep cells in fixed states, while in vivo cells may adapt transient and reversible states. Here, we have tested the existence and role of epithelial-mesenchymal plasticity in metastasis of mammary tumors without artificially modifying EMT regulators. In these tumors, we found by intravital microscopy that the motile tumor cells have undergone EMT, while their epithelial counterparts were not migratory. Moreover, we found that epithelial-mesenchymal plasticity renders any EMT-induced stemness differences, as reported previously, irrelevant for metastatic outgrowth, because mesenchymal cells that arrive at secondary sites convert to the epithelial state within one or two divisions, thereby obtaining the same stem cell potential as their arrived epithelial counterparts. We conclude that epithelial-mesenchymal plasticity supports migration but additionally eliminates stemness-enhanced metastatic outgrowth differences.

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Extracellular vesicle (EV) transfer is increasingly recognized as an important mode of intercellular communication by transferring a wide variety of biomolecules between cells. The characterization of in vitro- or ex vivo-isolated EVs has considerably contributed to the understanding of biological functions of EV transfer. However, the study of EV release and uptake in an in vivo setting has remained challenging, because cells that take up EVs could not be discriminated from cells that do not take up EVs. Recently, a technique based on the Cre-loxP system was developed to fluorescently mark Cre-reporter cells that take up EVs released by Cre recombinase-expressing cells in various in vitro and in vivo settings. Here we describe a detailed protocol for the generation of Cre(+) cells and reporter(+) cells, which takes ∼ 6 weeks, and subsequent assays with these lines to study functional EV transfer in in vitro and in vivo (mouse) settings, which take up to ∼ 2 months.

Inhibition of active MMP9 early during tumorigenesis suppresses tumor cell migration, invasion, and colony formation and tilts the balance towards anti-tumor immunity by activating CD8+ T cells.

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Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Currently there is no cure for a metastatic disease and it is therefore critical to target the early events that foster metastasis. It is now also recognized that a favorable microenvironment in the metastatic site, primed by the tumor, is crucial for metastasis. Our study is geared towards deciphering cellular and molecular mechanisms governing the metastatic niche that may lead to novel targeted anti-metastatic therapeutics. We utilize the multi-stage MMTV-PyMT breast cancer mouse model, which shares significant similarities with human breast cancer. By injecting a reporter metastatic cell line into hyperplasia-bearing mice, we were able to probe the susceptibility of the lung microenvironment to metastatic seeding. We demonstrate that early during mammary tumorigenesis, before metastasis has occurred, a metastatic niche is formed in the lung microenvironment. This niche is initiated in part by tumor-induced systemic pro-inflammatory factors and local extracellular matrix remodelers, as matrix metalloproteinases (MMPs). We show that the metastatic niche is associated with MMP9-expressing CD11b+Gr1+ and other lung stromal cells. MMP9, which is also expressed by tumor cells, appears as a pivotal player in the process and is therefore considered as a desirable therapeutic target. To examine the role of MMP9 activity in lung metastatic colonization, we utilized novel endogenous-like, function-specific antibodies (SDS3) that block the transiently-activated enzyme conformation of MMP9, which presumably contributes to disease progression. The therapeutic potential of SDS3 has been demonstrated in models of inflammatory bowel disease. We show that metastatic seeding within the lung microenvironment can be inhibited by SDS3, not only in experimental metastasis models but also when the lung microenvironment is primed by hyperplastic mammary tumors. Primary tumor burden was not changed with SDS3, suggesting that blocking active MMP9 is effective in preventing early metastasis rather than established tumors. To study the biodistribution and pharmacokinetics of SDS3, we utilized whole-body bioluminescence, intravital and ex-vivo live microscopy as well as flow cytometry. We show that SDS3 is retained in myeloid cells within the microenvironment of mammary tumors and lung metastatic foci. In situ zymography shows high MMP activity in premetastaic MMTV-PyMT lungs, which is reduced after SDS3. SDS3 also inhibits colony formation of cultured metastatic cells. Our results suggest that a metastatic niche is present in the lungs of hyperplastic mammary tumor-bearing mice and that it can be targeted by blocking MMP9 activity. Our study offers new insights into effectively blocking the in vivo activity of dysregulated MMPs as early anti-metastatic therapy of various cancers. Citation Format: Vicki Plaks, Jonathan Chou, Carrie Maynard, Nguyen H. Nguyen, Niwen Kong, Inna Solomonov, Dalit Talmi-Frank, Caroline Bonnans, Irit Sagi, Zena Werb. Targeting matrix metalloproteinases (MMP) for anti-metastatic therapy: Blocking active MMP9 abrogates metastatic niche formation and prevents metastatic seeding in a breast cancer model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4724. doi:10.1158/1538-7445.AM2015-4724