Ayuko Hoshino

Tumour exosome integrins determine organotropic metastasis.

Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90–120 nm; small exosome vesicles, Exo-S, 60–80 nm) and discovered an abundant population of non-membranous nanoparticles termed ‘exomeres’ (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N-glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.Lyden and colleagues use asymmetric flow field-flow fractionation to classify nanoparticles derived from cell lines and human samples, including previously uncharacterized large, Exo-L and small, Exo-S, exosome subsets.

Complex polymorphisms in endocytosis genes suggest alpha-cyclodextrin as a treatment for breast cancer

Most breast cancer deaths are caused by metastasis and treatment options beyond radiation and cytotoxic drugs, which have severe side effects, and hormonal treatments, which are or become ineffective for many patients, are urgently needed. This study reanalyzed existing data from three genome-wide association studies (GWAS) using a novel computational biostatistics approach (muGWAS), which had been validated in studies of 600–2000 subjects in epilepsy and autism. MuGWAS jointly analyzes several neighboring single nucleotide polymorphisms while incorporating knowledge about genetics of heritable diseases into the statistical method and about GWAS into the rules for determining adaptive genome-wide significance. Results from three independent GWAS of 1000–2000 subjects each, which were made available under the National Institute of Health’s “Up For A Challenge” (U4C) project, not only confirmed cell-cycle control and receptor/AKT signaling, but, for the first time in breast cancer GWAS, also consistently identified many genes involved in endo-/exocytosis (EEC), most of which had already been observed in functional and expression studies of breast cancer. In particular, the findings include genes that translocate (ATP8A1, ATP8B1, ANO4, ABCA1) and metabolize (AGPAT3, AGPAT4, DGKQ, LPPR1) phospholipids entering the phosphatidylinositol cycle, which controls EEC. These novel findings suggest scavenging phospholipids as a novel intervention to control local spread of cancer, packaging of exosomes (which prepare distant microenvironment for organ-specific metastases), and endocytosis of β1 integrins (which are required for spread of metastatic phenotype and mesenchymal migration of tumor cells). Beta-cyclodextrins (βCD) have already been shown to be effective in in vitro and animal studies of breast cancer, but exhibits cholesterol-related ototoxicity. The smaller alpha-cyclodextrins (αCD) also scavenges phospholipids, but cannot fit cholesterol. An in-vitro study presented here confirms hydroxypropyl (HP)-αCD to be twice as effective as HPβCD against migration of human cells of both receptor negative and estrogen-receptor positive breast cancer. If the previous successful animal studies with βCDs are replicated with the safer and more effective αCDs, clinical trials of adjuvant treatment with αCDs are warranted. Ultimately, all breast cancer are expected to benefit from treatment with HPαCD, but women with triple-negative breast cancer (TNBC) will benefit most, because they have fewer treatment options and their cancer advances more aggressively.

Complex polymorphisms in endocytosis genes suggest alpha-cyclodextrin against metastases in breast cancer

Most breast cancer deaths are caused by metastasis and treatment options beyond radiation and cytotoxic drugs, which have severe side effects, and hormonal treatments, which are or become ineffective for many patients, are urgently needed. This study reanalyzed existing data from three genome-wide association studies (GWAS) using a novel computational biostatistics approach (muGWAS), which had been validated in studies of 600–2000 subjects in epilepsy and autism. MuGWAS jointly analyzes several neighboring single nucleotide polymorphisms while incorporating knowledge about genetics of heritable diseases into the statistical method and about GWAS into the rules for determining adaptive genome-wide significance. Results from three independent GWAS of 1000–2000 subjects each, which were made available under the National Institute of Health’s “Up For A Challenge” (U4C) project, not only confirmed cell-cycle control and receptor/AKT signaling, but, for the first time in breast cancer GWAS, also consistently identified many genes involved in endo-/exocytosis (EEC), most of which had already been observed in functional and expression studies of breast cancer. In particular, the findings include genes that translocate (ATP8A1, ATP8B1, ANO4, ABCA1) and metabolize (AGPAT3, AGPAT4, DGKQ, LPPR1) phospholipids entering the phosphatidylinositol cycle, which controls EEC. These novel findings suggest scavenging phospholipids via alpha-cyclodextrins (αCD) as a novel intervention to control local spread of cancer, packaging of exosomes (which prepare distant microenvironment for organ-specific metastases), and endocytosis of β1 integrins (which are required for spread of metastatic phenotype and mesenchymal migration of tumor cells). Beta-cyclodextrins (βCD) have already been shown to be effective in in vitro and animal studies of breast cancer, but exhibits cholesterol-related ototoxicity. The smaller αCDs also scavenges phospholipids, but cannot fit cholesterol. An in-vitro study presented here confirms hydroxypropyl (HP)-αCD to be twice as effective as HPβCD against migration of human cells of both receptor negative and estrogen-receptor positive breast cancer. If the previous successful animal studies with βCDs are replicated with the safer and more effective αCDs, clinical trials of adjuvant treatment with αCDs are warranted in women with triple-negative breast cancer, who progress fast and have few treatment options.

A Freeze Drying Sample Preparation Method for Correlative Light and Scanning/Transmission Electron Microscopy

1. Electron Microscopy Resource Center, The Rockefeller University, New York, NY, USA. 2. Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, USA 3. Department of Infectious Diseases, King’s College London, London, United Kingdom 4. Children’s Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children’s Health, Meyer Cancer Center, Weill Cornell Medicine, New York, New York, USA.

Abstract 4805: The role of exosomes in fibrosarcoma progression

Among pediatric cancers, sarcomas, especially those with large tumor burdens and metastatic disease, often result in poor outcome. Thus, new treatments are urgently needed to inhibit tumor progression, prevent metastasis, and improve overall survival. To understand the mechanisms driving sarcoma progression, we employed two mouse fibrosarcoma cell lines that display different growth phenotypes when transplanted into syngeneic immune competent mice. Progressor fibrosarcomas evade detection by the immune system and develop large tumor burdens, while regressor fibrosarcomas regress shortly after a period of limited tumor growth. This difference in the growth phenotype is mediated in part by immune cells, but the mechanisms by which progressor and regressor cells influence immune cell activity are not fully elucidated. Our research has focused on exosomes, 50-100 nm secreted nanovesicles, that contain bioactive cargoes and have emerged as mediators of intercellular communication between various cells. Here, we hypothesize that fibrosarcoma exosomes determine the aggressiveness of the disease by either educating tumor cells themselves or altering interactions with host cells. To investigate the role of fibrosarcoma exosomes, we first educated regressor tumor cells with progressor exosomes for 3 weeks in vitro, and vice versa. Upon in vivo inoculation, progressor cells conditioned with regressor cell-derived exosomes grew significantly slower compared to untreated progressor cells. In contrast, regressor cells educated with progressor exosomes did not regress and overgrew significantly compared to untreated regressor cells. These results indicate that exosomes have an ability to reprogram a tumor phenotype. To determine if fibrosarcoma exosomes specifically impact the host, we evaluated whether conditioning naive mice with progressor exosomes alters in vivo growth of regressor tumor cells injected after three weeks of education. Twenty percent of regressor tumors continued to grow following progressor exosome education, while all tumors regressed in the control PBS group. These data suggest that exosomes also play an important role in the interaction with the host cells influencing tumor growth and disease progression. Next, to determine which exosomal cargo may mediate these effects, we sought to characterize the proteome of exosomes from progressor and regressor cells using mass spectrometry. We found basigin, also known as CD147, a transmembrane embryonic glycoprotein reported to regulate cell proliferation, tumor migration and metastasis as one of the candidate proteins highly expressed in progressor-derived exosomes. We conclude that exosomes derived from fibrosarcoma progresssors and regressors can alter the tumor phenotype both directly and indirectly. Exosomal cargo, such as basigin, may mediate the aggressive behavior of tumor cells, making it a potential therapeutic target to inhibit fibrosarcoma progression. Citation Format: Miho Nakajima, Stephen C. Searles, Ayuko Hoshino, Katherine M. Offer, Candia M. Kenific, Jack D. Bui, David C. Lyden. The role of exosomes in fibrosarcoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4805. doi:10.1158/1538-7445.AM2017-4805