Implications of Extracellular Vesicle Transfer on Cellular Heterogeneity in Cancer: What Are the Potential Clinical Ramifications?

The functional and phenotypic heterogeneity of tumor cells represents one of the greatest challenges in the successful treatment of cancer patients, because it increases the risk that certain individual tumor cells possess the ability to, for example, metastasize or to tolerate cytotoxic drugs. This heterogeneity in cellular behavior is driven by genetic and epigenetic changes and environmental differences. Recent studies suggest that an additional layer of complexity of tumor heterogeneity exists, based on the ability of cells to share functional biomolecules through local and systemic transfer of extracellular vesicles (EV), with profound effects on cellular behavior. The transfer of functional biomolecules between various populations of tumor cells and between tumor cells and nontumor cells has large consequences for both the tumor cells and the microenvironment that support the cellular behavior of tumor cells, and therefore for the clinical outcome of cancer. Here, we discuss the latest findings on EV transfer and the potential implications of EV-mediated local and systemic transmission of phenotypic behavior, particularly in the context of tumor heterogeneity, metastatic disease, and treatment response. Cancer Res; 76(8); 2071-5. ©2016 AACR

Cranial imaging window implantation technique for longitudinal multimodal imaging of the brain environment in live mice

Summary: Intravital two-photon microscopy of the mouse brain requires visual access without affecting normal cognitive functions, which is crucial for longitudinal imaging studies that may last several months. In this protocol, we describe the surgical implantation of a metal-free cranial imaging window, which can be used to perform two-photon microscopy and magnetic resonance imaging in the same animal. This multimodal imaging platform enables the investigation of dynamic processes in the central nervous system at a cellular and macroscopic level.For complete details on the use and execution of this protocol in the context of brain cancer, please refer to Zomer et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Studying extracellular vesicle transfer by a Cre-loxP method

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

Exosomes: Fit to deliver small RNA

Exosomes are specialized membranous nano-sized vesicles derived from endocytic compartments that are released by many cell types. Microvesicles are distinctive from exosomes in that they are produced by shedding of the plasmamembrane and usually larger in size (>1 µm). Exosome biogenesis involves the tightly controlled process of inward budding from the limiting membrane of multivesicular bodies (MVBs). This results in numerous intraluminal vesicles in the lumen of MVBs that contain distinct protein repertoires. It has been suggested that microvesicles shed by certain tumor cells hold functional messenger RNA (mRNA) that may promote tumor progression. We discovered that purified exosomes contain functional microRNAs (miRNAs) and small RNA, but detected little mRNA. Although a clear and decisive distinction between microvesicles and exosomes cannot be made and different subsets of exosomes exist, we speculate that exosomes are specialized in carrying small RNA including the class 22–25 nucleotide regulatory miRNAs. To demonstrate this we developed a co-culture system and found that exosomes are continuously secreted and transferred from Epstein Barr virus (EBV)-infected cells to uninfected neighboring cells. Throughout exosome transfer, the exogenous EBV-encoded miRNAs were delivered to subcellular sites of miRNA-mediated gene repression. Additionally, we found evidence that mature miRNAs are transferred between circulating cells in humans, since we detected EBV-miRNAs in non-infected cells in the peripheral blood of patients that include monocytes and T cells. In this addendum we discuss these findings in the context of recently published papers that advanced our current knowledge of exosome physiology, (mi)RNA function and intercellular RNA transfer. Based on this information we propose that an intercellular (miRNA-based) mode of signal transmission may be well suited in controlling space-confined processes such as the initiation of immune responses in the secondary (peripheral) lymphoid tissues or in a tumor microenvironment. Deciphering the molecular mechanism(s) that control small RNA loading into exosomes and transfer to recipient cells in vitro will provide new evidence for the physiological relevance of vesicle-mediated intercellular communication in vivo

Identity and dynamics of mammary stem cells during branching morphogenesis

During puberty, the mouse mammary gland develops into a highly branched epithelial network. Owing to the absence of exclusive stem cell markers, the location, multiplicity, dynamics and fate of mammary stem cells (MaSCs), which drive branching morphogenesis, are unknown. Here we show that morphogenesis is driven by proliferative terminal end buds that terminate or bifurcate witwh near equal probability, in a stochastic and time-invariant manner, leading to a heterogeneous epithelial network. We show that the majority of terminal end bud cells function as highly proliferative, lineage-committed MaSCs that are heterogeneous in their expression profile and short-term contribution to ductal extension. Yet, through cell rearrangements during terminal end bud bifurcation, each MaSC is able to contribute actively to long-term growth. Our study shows that the behaviour of MaSCs is not directly linked to a single expression profile. Instead, morphogenesis relies upon lineage-restricted heterogeneous MaSC populations that function as single equipotent pools in the long term

In Vivo Imaging Reveals Extracellular Vesicle-Mediated Phenocopying of Metastatic Behavior

Most 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

Cancer cells copy migratory behavior and exchange signaling networks via extracellular vesicles

Recent data showed that cancer cells from different tumor subtypes with distinct metastatic potential influence each other's metastatic behavior by exchanging biomolecules through extracellular vesicles (EVs). However, it is debated how small amounts of cargo can mediate this effect, especially in tumors where all cells are from one subtype, and only subtle molecular differences drive metastatic heterogeneity. To study this, we have characterized the content of EVs shed in vivo by two clones of melanoma (B16) tumors with distinct metastatic potential. Using the Cre-LoxP system and intravital microscopy, we show that cells from these distinct clones phenocopy their migratory behavior through EV exchange. By tandem mass spectrometry and RNA sequencing, we show that EVs shed by these clones into the tumor microenvironment contain thousands of different proteins and RNAs, and many of these biomolecules are from interconnected signaling networks involved in cellular processes such as migration. Thus, EVs contain numerous proteins and RNAs and act on recipient cells by invoking a multi-faceted biological response including cell migration

Cancer cells copy migratory behavior and exchange signaling networks via extracellular vesicles

Recent data showed that cancer cells from different tumor subtypes with distinct metastatic potential influence each other's metastatic behavior by exchanging biomolecules through extracellular vesicles (EVs). However, it is debated how small amounts of cargo can mediate this effect, especially in tumors where all cells are from one subtype, and only subtle molecular differences drive metastatic heterogeneity. To study this, we have characterized the content of EVs shed in vivo by two clones of melanoma (B16) tumors with distinct metastatic potential. Using the Cre-LoxP system and intravital microscopy, we show that cells from these distinct clones phenocopy their migratory behavior through EV exchange. By tandem mass spectrometry and RNA sequencing, we show that EVs shed by these clones into the tumor microenvironment contain thousands of different proteins and RNAs, and many of these biomolecules are from interconnected signaling networks involved in cellular processes such as migration. Thus, EVs contain numerous proteins and RNAs and act on recipient cells by invoking a multi-faceted biological response including cell migration

Studying extracellular vesicle transfer by a Cre-loxP method

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