Alissa M. Weaver - US grants

DESCRIPTION (provided by applicant): The objective of this application is to develop the career of Dr. Alissa Weaver as an independent investigator in the field of cancer invasion and metastasis. Cortactin is a prominent src kinase substrate that is overexpressed in 13% of breast cancers via 11q13 amplification, a chromosomal change associated with increased tumor aggressiveness and poor patient prognosis. The nucleation of new actin filaments by the Arp2/3 complex is essential for protrusion of the leading edge of migrating cells and also contributes to the creation of additional subcellular structures, such as cancer cell invadopodia. Previous studies by this investigator have demonstrated that cortactin promotes actin assembly by the Arp2/3 complex and works in concert with N-WASp, another Arp2/3 activator and src substrate. Both cortactin and N-WASp have been implicated in cancer cell invadopodia formation, but the mechanism of recruitment and action is unclear. The goal of this proposal is to test the hypothesis that cortactin functions as an integrator of signals to the cytoskeleton and controls critical steps in breast cancer invasion and metastasis. Three specific aims are proposed: In Specific Aim 1, we will test the hypothesis that a key function of cortactin is to assemble signaling and cytoskeletal proteins, including src kinase and N-WASp, for actin assembly in breast cancer cell invadopodia and lamellipodia. In Specific Aim 2, we will determine the role of cortactin and N-WASp in the morphologic and phenotypic changes that characterize the epithelial-mesenchymal transition. In Specific Aim 3, we will develop a tetracycline-inducible transgenic mouse model to test the hypothesis that cortactin promotes branching morphogenesis of the mammary gland and breast cancer metastasis. We anticipate that these studies will yield important insight into the mechanisms by which metastasis occurs in vivo and potentially allow the identification of novel cytoskeleton-based targets for rational drug design.

[unreadable] DESCRIPTION (provided by applicant): This proposal examines mechanisms of cell motility at a fundamental level. We focus on clarifying the relationship between lamellipodial dynamics and focal complex formation, at the leading edge of a migrating cell. We are in a unique position to conduct these studies at the molecular level, because our published and preliminary data have unveiled a core mechanism whereby cortactin couples lamellipodial persistence with focal complex assembly. In Aim 1 we will test the hypothesis that this coupling is in effect akin to a "rack and pinion" steering mechanism for moving cells, which allows for a dominant lamellipodia to form and guide the cell in its direction. In Aim 2 we will define how lamellipodia persistence is generated and positively regulated. Our existing data already conclusively show that binding of cortactin to branched actin is the basis for persistence, and we will test whether cortactin stabilizes branches, super-activates Arp2/3 complex, or both. In Aim 3 we will define how persistence is spatially constrained and negatively regulated at areas of lamellipodia activity, by investigating molecular mechanisms of cortactin inhibition by phospholipids and cofilin, at the front and rear of the lamellipodia, respectively. An essential aspect of our studies is that we combine biochemistry with quantitative microscopy in order to determine not only whether molecular mechanisms can happen (e.g., in a test tube with purified components), but also whether in fact they occur in a living cell. These approaches include: chemotaxis and other cell motility assays, quantitative analyses of lamellipodial and adhesion dynamics in living cells, and careful biochemical characterization of actin binding protein mutants followed by quantification of cell phenotypes they produce. With the proposed studies on lamellipodial persistence, we are positioning ourselves at the interface of the adhesion and actin biology fields, whose integration will hopefully generate a new exciting discipline. Significance: Our studies are significant both for fundamental cell biology and human health. The molecular mechanisms we are attempting to solve are not only critical for cell motility, but also for the many cellular functions that depend on branched actin assembly, including vesicular trafficking and tissue morphogenesis. With respect to human health, these studies are particularly relevant to cancer metastasis, since cell motility is an essential component of cancer cell invasion. More directly, cortactin is well- documented to be overexpressed in a number of cancers via gene amplification, including 15% of breast and 30% of head and neck squamous cell cancer (HNSCC). Intriguingly, cortactin overexpression correlates with poor prognosis and decreased survival. Thus, the studies in this proposal are important for understanding both the fundamental regulation of dynamic branched actin assemblies and the possible role of cortactin specifically in cancer cell motility. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with 40,000 new cases and 11,000 deaths occurring each year in the United States. Despite advances that improve the quality of life for patients, there has been little to no improvement in survival for the last 30 years. Among the genetic aberrations that occur in HNSCC, 11q13 amplification is one of the most common and is strongly linked to poor prognosis and decreased survival of patients. A strong candidate gene within that amplicon to promote tumor aggressiveness is cortactin, a cytoskeletal protein that promotes cell motility and invasion. Mechanistically, cortactin is an essential component of invadopodia, cell surface protrusions thought to be involved in cancer invasion; however it is not clear to what extent this activity contributes to tumor growth and aggressiveness in vivo. The goal of our study is to test the individual role of cortactin in progression of 11q13-amplified HNSCC tumors. Through the use of retroviral expression systems to manipulate cortactin expression in non-amplified and 11q13-amplified HNSCC cell lines, we will test the role of cortactin in HNSCC tumor growth and invasion in vivo and invadopodia activity in vitro. By the end of the grant period, we should have determined 1) whether cortactin promotes HNSCC tumor progression and thus represents a good target for treatment; and 2) whether there is a more general link between invadopodia activity and tumor growth. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): In humans, a common risk factor for the development of invasive breast cancer is dense breast tissue, detected by mammography. This dense tissue is associated with increased stromal collagen, increased epithelial cells, and decreased fatty tissue. Recent studies in transgenic mice indicate that increased collagen density in breast stromal tissue plays a causative role in promoting both the formation and invasiveness of breast tumors. Additional studies implicate tissue rigidity downstream of extracellular matrix (ECM) deposition and/or cross linking in promoting aggressive, invasive cellular phenotypes. Conversely, basement membrane ECM proteins that underlie normal and carcinoma in situ epithelial cells are thought to inhibit tumor progression. Because of the complexity of extracellular matrix-tumor cell interactions, it is difficult to fully isolate and understand the various effects of extracellular matrix on tumor progression using traditional biological approaches. We therefore propose to use an interdisciplinary approach in which we develop a 3-dimensional cell-based multiscale mathematical model of ECM-breast cancer interactions. Using this model, we will perform in silico experiments to test the overall hypothesis that ECM rigidity and stromal collagen fibrosis provide an environment that promotes tumor progression and invasion. We will specifically test the role of collagen fibril density, width, alignment, and crosslinking and the role of basement membrane ECM and proteases on cancer growth and invasion. All modeling will be fully integrated with experimentation to obtain realistic parameters and separately test predictions. We anticipate that this project will identify critical microenvironmental factors promoting breast cancer progression and lay the groundwork for future therapeutic intervention.

DESCRIPTION (provided by applicant): Recent studies have identified tumor-derived microvesicles called exosomes as vehicles for long-distance communication, due to their complex content of proteins and microRNAs. In head and neck squamous cell carcinoma, as in many other cancers, exosome secretion is associated with advanced patient stage. In most cases, those vesicles are classified as exosomes, 50-100 nm vesicles that have been shown to mediate progression, metastasis, survival, drug resistance, immune modulation, and many other aggressive cancer phenotypes. The mechanisms by which exosomes are generated are poorly understood, although exosomes are known to derive from a late endocytic compartment. Our recent preliminary data suggest that invadopodia are sites of exosome secretion and that, conversely, canonical invadopodia regulators affect exosome secretion. Based on these and other findings, we hypothesize that exocytic late endosomal/lysosomal pathways that govern invadopodia activity and exosome secretion may be one and the same. Furthermore, these pathways are likely to be unregulated in the 30-40% of HNSCC tumors that carry amplification of the 11q13.3 amplicon, since we have shown that the 11q13-amplified cytoskeletal protein cortactin is a key regulator of invadopodia activity, exosome secretion, and tumor aggressiveness. In this project, we will test whether invadopodia represent docking sites for exosomes and identify key intracellular regulatory points for exosome secretion by HNSCC cells. We will also test the hypothesis that 11q13-amplification is an independent predictor of exosome secretion in HNSCC patients. Finally, we will determine whether inhibition of exosome secretion represents a viable therapeutic strategy in HNSCC.

? DESCRIPTION (provided by applicant): Secretion of extracellular exosome vesicles from cells can have profound autocrine and paracrine effects on cellular phenotype. In the tumor microenvironment, exosomes are thought to be important mediators of tumor progression. However, the exact functions of exosomes in tumors are poorly understood. Although not initially thought to be specific exosome cargos, extracellular matrix molecules are frequently identified in association with exosomes and could profoundly affect tumor behavior. The Weaver laboratory recently demonstrated that the matrix molecule fibronectin is specifically targeted to exosomes by interacting with integrin receptors. Furthermore, they have found that secretion of fibronectin-carrying exosomes promotes adhesion formation and cancer cell motility. Finally, their preliminary data indicates that exosome secretion is also important for stromal matrix assembly and alignment, which the Keely lab has demonstrated is an important driver of poor prognosis in breast cancer. Based on these data, the central hypothesis of this project is that both autocrine and paracrine communication via fibroblast and breast cancer exosome secretion drives generation of aligned stromal matrices and aggressive tumor behavior. To test this hypothesis, we will determine the role of exosomes in driving persistent cell migration by breast cancer cells. We will also test the hypothesis that autocrine exosome secretion is critical for stromal matrix assembly and alignment by fibroblasts. Finally, we will define the role of breast cancer- fibroblast exosomal crosstalk in promoting stromal matrix assembly and tumor aggressiveness.

? DESCRIPTION (provided by applicant): Exosomes are small late endosome-derived extracellular vesicles that carry bioactive protein and RNA molecules and modulate cell behavior. Recent data indicate that a wide variety of cells, including cancer cells and neurons, secrete exosomes. Furthermore, preliminary reports indicate that secreted exosomes critically contribute to several physiological processes, including invasion and motility, stem cell fate, and angiogenesis. However, the specific biological effects of exosomes on cells are poorly understood. We recently identified invasive actin-based protrusions called invadopodia as specific docking sites for exosome-containing multivesicular endosomes (MVE) and showed that exosome secretion controls invadopodia biogenesis and activity. Our further investigations have uncovered a fundamental role for exosomes in regulating the formation of additional related actin-based motility structures: adhesions, and filopodia. Exosomes appear to have a particularly potent effect on filopodia, which are adhesive actin-based protrusions that are important for directional sensing, cell polarity, cell movement, and cellular interactions. We therefore propose the central hypothesis that a key function of exosomes is to promote formation of filopodia and thereby control local cellular behaviors. We will test this hypothesis i both cancer cells and neurons because these cells exhibit robust filopodia and filopodia control key cellular behaviors. We will identify the temporal and functional relationship between exosome secretion and filopodia formation. We will identify molecular exosome cargos that control filopodia formation and maturation and determine the mechanisms by which they act. Finally, we will determine the role of exosomes in regulating complex filopodia-dependent processes, including synapse formation and directed migration both in vitro and in vivo.

PROJECT SUMMARY ? PROJECT 2 Small Cell Lung Carcinoma (SCLC) is an aggressive neuroendocrine subtype of lung cancer. SCLC patients have a very low 5-year survival, in large part because SCLC tumors can become rapidly resistant to chemotherapy and radiation therapy and because of a lack of targeted therapies. Emerging evidence supports the idea that, while SCLC tumors seem homogeneous when examined under a microscope, these tumors contain a significant level of intra-tumoral heterogeneity. Indeed, recent observations by our group and others have identified distinct cellular phenotypes in SCLC, including in primary human tumors, in cell lines derived from human tumors, and in tumors from genetically-engineered mouse models. Importantly, data from our group as well as from Project 1 investigators indicate that these cellular phenotypes contribute to SCLC development and potentially response to therapy. The specific goal of this proposal is to elucidate how different cellular subpopulations within SCLC tumors drive SCLC dynamics, growth, survival, and aggressiveness as an ecosystem. To accomplish this goal, we will focus on better understanding the nature of SCLC phenotypic subtypes and how these populations functionally interact with each other and with noncancerous cells in the tumor microenvironment. Specifically, we have previously identified stem-like tumor-propagating cells (TPCs) in SCLC tumors and found that these cells are neuroendocrine and strongly tumorigenic. We have also characterized cell populations derived from these TPCs with distinct phenotypes, including non-neuroendocrine subpopulations that can promote the growth and the spread of the neuroendocrine TPCs. Leveraging these findings as well as our unique genetic mouse models that allow dissection of SCLC phenotype evolution, we will use a combination of experimental and mathematical approaches to investigate how these different SCLC cell types contribute to tumor growth, in relationship with the tumor microenvironment. We will build and use mathematical modeling to predict key interactions between SCLC subpopulations with distinct phenotypes and to uncover fragility/intervention points that could be used for treatment. Modeling will also be a key factor driving experimental design. As part of this design, we will determine how cell-cell interactions in SCLC tumors affect the division and survival rates of the different subpopulations; we will also determine phenotype transition rates between different subpopulations to capture SCLC dynamics and plasticity. Finally, we will elucidate the role of secretory factors released by these SCLC subpopulations in driving survival, growth, phenotype composition, and metastasis of SCLC tumors. These experiments will elucidate basic mechanisms of SCLC development and progression and may ultimately lead to novel therapeutic approaches by identifying key interactions of SCLC subpopulations.

A project summary/abstract is not required for this section.

PROJECT SUMMARY Small Cell Lung Carcinoma (SCLC) is an aggressive neuroendocrine subtype of lung cancer. SCLC patients have a very low 5-year survival, in part because SCLC tumors are often detected at a late stage when the tumors have already metastasized and treatment outcomes are worse. Thus, early detection becomes critical to achieve better treatment results. Emerging evidence supports the idea that, while SCLC tumors seem homogeneous when examined under a microscope, these tumors contain a significant level of intra-tumoral heterogeneity. Indeed, recent observations by our group and others have identified distinct cellular phenotypes in SCLC, including in primary human tumors, in cell lines derived from human tumors, and in tumors from genetically-engineered mouse models. Importantly, data from our group indicate that these cellular phenotypes contribute to SCLC development. The specific goal of this proposal is to elucidate how different cellular subpopulations within SCLC tumors drive early SCLC development, dynamics, and growth and to leverage this mechanistic information to identify biomarkers for early detection and prevention of SCLC. We have previously identified tumor-propagating cells (TPCs) in SCLC tumors and found that these cells are neuroendocrine and strongly tumorigenic. We have also characterized cell populations derived from these TPCs with distinct phenotypes, including non-neuroendocrine NOTCH+ and CD44+ subpopulations, that promote the growth and survival of the neuroendocrine TPCs. Leveraging these findings as well as our unique genetic mouse models that allow dissection of SCLC phenotype evolution, we will investigate how cell-cell interactions of these distinct SCLC cell phenotypes contribute to tumor development and growth, in relationship with the tumor microenvironment. We will also elucidate the role of secretory factors released by these SCLC subpopulations in driving survival, growth, and phenotype composition of SCLC tumors. Finally, we will perform analysis of cfDNA and proteins (including on exosomes) present in SCLC patient plasma for identification of related markers of SCLC development and early detection. We will also follow up on intriguing findings that germline mutations in NOTCH are present in a large fraction of SCLC patients, suggesting a potential risk marker beyond smoking. This interdisciplinary basic-translational project will elucidate fundamental mechanisms of SCLC development and may lead to novel methods for early detection and/or prevention of SCLC.

Project 1 Summary: Extracellular vesicles (EVs) carry a variety of RNAs, including both coding and noncoding RNAs. These RNAs have the potential to influence cell and tissue phenotypes. Indeed for miRNAs there are now many examples of EV-carried miRNA controlling gene expression and function in recipient cells. RNA-Seq analyses have demonstrated specific enrichment of some RNAs in EVs, compared to the cellular content. However, very little is known about the specific mechanisms by which RNA is transported into EVs. The current paradigm, based on several studies, is that RNA-binding proteins (RBPs) are responsible for the selective and specific inclusion of RNAs in EVs. However, how those RBPs connect to cellular membranes to be incorporated into shed microvesicles (MVs) or late endosome-derived exosomes is unknown. A notable finding is that many RBPs identified in EVs are typically associated with the endoplasmic reticulum (ER) in cells, suggesting a potential role for the ER in transfer of those moieties to other organelles. Furthermore, the RNA-induced silencing complex is assembled on mRNA-ribosome complexes associated with the ER (rough ER), suggesting a route for miRNA-RBP association with the ER and subsequently other membranes. Based on these findings and our preliminary data, we hypothesize that ER-plasma membrane (PM) and ER-multivesicular endosome (MVE) membrane contact sites (MCS) are critical for transfer of RNAs and RBPs into shed microvesicles and exosomes. We further hypothesize that signaling and lipid transfer events taking place at these contacts further regulate RNA transport into vesicles. We will test these hypotheses and determine the impact of MCS on transfer of RNAs to recipient cells and CRC tumor growth and cetuximab resistance.

Administrative Core Summary Effective administration is critical to the mission of the Program. The major goals of the Administrative Core are to coordinate and direct Program efforts, to interface with other leaders/efforts in the field, and to interface with NCI. As part of this effort, the Administrative Core will also organize outside speaker visits, scientific retreats, and the annual External Advisory Committee (EAC) meetings. The Administrative Core will also monitor progress in the Program and effective use of the Shared Resource Cores. To achieve these goals, the Administrative Core will be led by the Program Principal Investigator, Dr. Alissa Weaver. Dr. Weaver will interface with all Core leaders at formal quarterly meetings, and all Project leaders at monthly meetings to review progress and determine what, if any, changes need to be made. In addition, every two weeks the entire Program investigator group will meet for data presentations. At those meetings, Projects and Cores will alternate presenting scientific results and we will also communicate any Program news or discuss any issues. Once a year, we will hold an Annual Research Retreat. A Program Administrator will facilitate organization and communication with Program members and outside investigators, including the EAC. The frequent meetings and formal structure that we will put in place will facilitate communication, allow early recognition of any problems that need to be addressed, allow capitalization on any opportunities, and ensure integration of the group and scientific synergy.

Abstract Metastasis is a well-known driver of cancer-related deaths. Nevertheless, limited success has been achieved in targeting cancer metastasis because it is an exceedingly complex process driven by multiple, integrated mechanisms. Collaborative studies in the Zijlstra and Weaver laboratories studied two separate aspects of cell motility: a) the dynamics of cell-cell adhesion controlled by proteolytic shedding of adhesion receptor and b) the release of motility promoting extracellular vesicles (EV). Since these two events take place in the same cells and contributed to the same phenotype, we speculated that these two biological processes were coordinated. Indeed, preliminary studies demonstrated that syntenin-1, a key component of the EV biogenesis pathways, was part of a cell adhesion complex anchored by the IgG superfamily member Activated Leukocyte Cell Adhesion Molecule (ALCAM) and its companion-tetraspanin CD151. Altering the expression and/or shedding of ALCAM drastically impacted EV biogenesis, confirming our original idea that cell-cell adhesion could be coordinated with EV biogenesis. The hypothesis that this occurred through an intracellular link between ALCAM and syntenin is further supported by the ability of free intracellular domain to suppress EV biogenesis. Based on these observations and our published expertise in cell adhesion, EV biology and metastasis, we propose to investigate the integration between cell-adhesion and the production of motility-promoting EVs during cancer progression. Specifically, the proposed studies will investigate: 1) the mechanistic integration between cell-adhesion and EV biogenesis, 2) the consequences for cargo incorporated in motility-promoting EVs, and 3) the functional contribution to autocrine and paracrine communication. Moreover, the relevance of this biology will be tested in the context of bladder cancer where ALCAM shedding is an independent prognostic indicator of survival. For this purpose we have developed a novel ex vivo organotypic culture system for bladder urothelium and bladder cancer in which we can replicate the clinical phenotypes of both papilloma and carcinoma of the bladder. Considering that tumor cells have a large number of divergent mechanisms at their disposal by which they can enhance their malignant behavior, determining how mechanisms of cell adhesion and EV biogenesis integrate is not only an innovative way to deconvolve complex metastatic behavior, it will also have significant clinical impact. With findings from the propose studies, will provide novel avenues of intervention where a therapy may target a point of synergy and integration rather than a direct mode of action.

The overall theme of ISEV2020, the annual meeting for the International Society for Extracellular Vesicles (ISEV) is ?Extracellular Vesicles: translating basic science to address human diseases?. Extracellular vesicles (EVs) are small lipid enclosed carriers of bioactive proteins, lipids, and nucleic acids. Only recently discovered, there is emerging recognition that EVs play a fundamental, critical, and evolutionarily conserved role in cellular communication. Because EVs are found in all body fluids, the EV profile of an individual may represent a snapshot of health and disease. While there has been a great deal of interest in this area, this is a very new field and the answers to many fundamental questions are outstanding. There is also a need to develop new technologies, in order to properly study EVs and harness their potential for clinical applications. Expected to be ~1400 participants, ISEV2020 will bring together the largest group of clinicians and basic scientists to exchange information and establish collaborations in EV research. No other meeting in the world offers the scope, participation level, and thematic focus of ISEV2020, cross-pollinating scientific investigations in the fields of disease biomarkers and therapeutic tools by disseminating cutting-edge developments in EV research. ISEV is also the premiere forum for disseminating information and establishing new efforts in the areas of Rigor and Reproducibility, Standardization, and EV characterization. The research theme includes diverse areas of science encompassing cancer, cardiovascular and renal disease, immunology, neurobiology and neurodegenerative diseases, diseases of aging, infectious disease, coagulation, rare and neglected diseases, environmental exposure, and vaccine development. Information provided at this meeting will be disseminated further through webcasting, ISEV websites and email blasts. We will also review key aspects of EV biology and technological developments at the Educational Day. The main goals are to disseminate the latest information on cutting edge discoveries and technologies, to further enhance Rigor and Standardization efforts, and to foster synergies between basic scientists and clinicians. Important goals of the meeting are to enhance participation from underrepresented minorities, minority institutions, and young investigators, and to expand cooperation between ISEV and the broader research community.

Extracellular vesicles (EVs) are cell-made particles that provide a natural mechanism of information and material transfer between cells. There is growing interest in large-scale production of EVs that can be used as therapeutics due to their ability to communicate signals from producer cells and their potential use as carriers for delivery of drug molecules. EV therapeutics are being developed for treatment of a wide range of diseases including metabolic disorders, cancer, and neurodegeneration. Despite the excitement generated by several early-stage EV biotech companies, the technology to produce mass quantities of purified EVs with tunable properties is still in its infancy. The long-term goal of this project is to enable production of "designer EVs" that can be packaged with desired cargo molecules, decorated with tunable surface ligands, and secreted in high yield from specific producer cell types. The overall objective of the current project is to identify cellular processes that can be engineered to control the production, content, and in vivo trafficking of therapeutic EVs. Making this new class of drugs available to the public has potential to improve the health and quality of life of millions of patients in the US and around the world. The project will also provide the unique educational opportunity for trainees to engage in collaborative research with industry scientists. Other broader impacts will be accomplished through engaging undergraduate and high-school students in EV research, and through integration of the project with the Vanderbilt Program for EV Research and bioengineering courses led by the PIs.

The investigators will focus their research program on loading and delivery of small RNAs, which are promising drug molecules that are not delivered efficiently to recipient cells using established nanoparticle-based carriers. The central hypothesis is that developing tissue-specific producer cells with tuned expression of EV-associated proteins and RNAs will enable researchers to maximize the product yield of specific EVs and the efficiency of RNA delivery from EVs to recipient cells. First, they will boost targeting of miRNAs to extracellular vesicles through modulating specific molecules at ER-contact sites. Second, they will optimize cargo delivery to target cells by engineering EVs for efficient endosomal escape and macrophage evasion properties. Third, they will develop a scalable platform for manufacturing tissue-specific EVs by differentiating induced pluripotent stem cells (iPSCs) to producer cells in 3D suspension cultures. The proposed research is innovative because it applies a multidisciplinary approach to address several critical barriers to commercial production of therapeutic EVs: cargo loading, cargo delivery, and scalable manufacturing. EVs secreted by MSCs are recognized as a viable alternative to overcome the potential risks from transplantation of primary MSCs. By developing the tools and strategies to customize and maximize EV production from iPSC-derived MSCs, the researchers will establish a flexible EV manufacturing platform that can expand to other relevant organ and tissue systems.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Summary ? Overall Component Cellular communication between diverse cells is now recognized to heavily influence both cancer initiation and progression. Recently, several new forms of intercellular communication have been recognized, including exchange of proteins and RNAs via extracellular vesicles (EVs) and other carriers. Extracellular RNA (ExRNA) is particularly interesting, as it has the potential to influence gene expression and potentially epigenetic states. MicroRNAs (miRNAs) in particular have been shown to be transferred from one cell type to another to influence gene expression; however, it is clear that many kinds of exRNAs are selectively secreted from cells. What is less clear is how those exRNAs (including miRNAs) are packaged into EVs and other carriers. In addition, the overall impact of these exRNAs on cells and tissues is not yet understood. In this program, we propose to elucidate the ?rules of the game? for extracellular RNA communication, using colorectal carcinoma (CRC) as a model system. Our Program will be highly synergistic, because each Project focuses on a different aspect of this problem. Thus, Project 1 will determine how subcellular contacts between organelles drive RNA and RNA-binding protein (RBP) transfer to EVs in CRC. Project 2 will identify RNA modifications and sequences that drive molecular selection of RNAs for transfer into EVs in CRC. Project 3 will identify how EVs and exomeres mediate EGFR-Wnt crosstalk in CRC. All Projects use common experimental systems, including CRC isogenic cell models, and have common biological focus (e.g. how miR-100 and miR-125b are trafficked and influence recipient cell function in CRC). Integration, synergy, and progress of the Projects will be highly enhanced by the proposed Administrative and Shared Resource Cores. Together this Program will make a major impact in the areas of CRC, tumor microenvironment, exRNA, EVs, and RNA biology.

SUMMARY - Core 2 The Extracellular Vesicle Purification and Analysis (EVPA) Core will uniquely serve the three individual projects within the Program Project Grant. Overall, the goal of the Core is centralize the methods, expertise, equipment, and skilled labor to perform extracellular vesicle (EV) purification, analysis, sizing, counting, and quality control. The Core will isolate EVs by differential centrifugation and density-gradient ultracentrifugation using Optiprep/ iodixanol layering. EVs will be counted and sized using nanoparticle tracking analysis. Moreover, this Core will perform Fluorescent Activated Vesicle Sorting (FAVS) which is based on fluorophore-conjugated antibodies to label EV surface markers and genetically encoded fluorescent markers to label internal proteins. This technique allows for the gating (counting) and sorting of EVs which are naturally below the diffraction limit of light based on size. EV preparations will further be sub-fractionated based on specialized density gradients to trace EV protein markers and RNA contents and isolate exomeres. For Project 1, this Core will purify EVs for all 3 Aims, as well as provide EV subfractionation and FAVS EV analysis and sorting for Aim 1C. For Project 2, this Core will purify EVs for all 3 Aims (1-3). For Project 3, this Core will utilize FAVS EV analysis and sorting and EV and exomere purification to achieve the goals of all 3 Aims (1-3). The EVPA Core will also engage in technology development to further develop the FAVS method to analyze single EVs based on fluorescent RNAs and RBPs, and to use fixed, permeabilized EVs for analysis. In summary, the goals of the Core are to provide uniform purification and analysis practices for all 3 projects and uniquely skilled technicians and equipment in a centralized Core. As such, this structure will facilitate extensive but timely characterization and delivery of high quality materials to investigators and drive the cutting edge in this area.

Exosomes are small extracellular vesicles (EVs) that are secreted from multivesicular endosomes (MVE) and have been recently recognized to promote cancer metastasis. Exosomes carry bioactive proteins, lipids and nucleic acids and are an important but poorly understood component of the tumor microenvironment. We recently discovered that actin-rich invasive structures called invadopodia are key docking sites for MVE in cancer cells, leading to enhanced exosome secretion. Furthermore, we found that the key invadopodia regulator cortactin enhances MVE docking and exosome secretion. Notably, cortactin is gene amplified and overexpressed in a number of cancers, especially in head and neck squamous cell carcinoma (HNSCC). Furthermore, cortactin overexpression in HNSCC is correlated with decreased patient survival and increased metastasis. Based on these data, we hypothesize that cortactin overexpression drives poor prognosis in HNSCC due to its key role in promoting exosome secretion. Furthermore, we hypothesize that key exosome cargoes synergize with cortactin to promote tumor-induced angiogenesis, lymphangiogenesis, and metastasis. Specifically, we have identified EphB-ephrinB signaling as a key angiogenic axis regulated by HNSCC-secreted exosomes. Thus, we propose that both the number and molecular cargo of exosomes drive aggressive HNSCC behavior in a synergistic manner. We will test these hypotheses and leverage our work to identify potential exosomal blood-and tissue-based biomarkers of regional and distant metastasis.