Browsing by Author "Kalluri, Raghu"
Now showing 1 - 15 of 15
Results Per Page
Sort Options
Item BIOGENESIS OF NATIVE EXTRACELLULAR VESICLES AND GENERATION OF BIOENGINEERED EXTRACELLULAR VESICLES AS THERAPEUTIC AGENTS(2024-10-02) Luo, Xin; Kalluri, Raghu; Grande-Allen, JaneExtracellular vesicles (EVs) are small vesicles secreted from presumably all types of body cells naturally. EVs are involved in the bidirectional intercellular communication with functional impact. While the mechanism of EV generation and uptake by recipient cells is not fully understood, which is crucial for understanding their biological impact, EVs have already been considered as a drug delivery system in the context of various pathologies. To better evaluate mechanism involved in the biogenesis of EVs, I focused on the functional role of three EV-enriched tetraspanins, CD9, CD63, and CD8. Employing loss of function studies, the proteomics of cells deficient in CD9, CD63, or CD81, and EVs generated by these cells were functionally investigated. CD9, CD63, and CD81 were found to be important for sorting of specific proteins into the EVs, each one displayed distinct contribution in trafficking of proteins into EVs . Next, to explore how engineered EVs can be involved in the regulation of immunity, I designed an engineered EV-based platform for vaccine development (EVX-M+P) and for cancer immunotherapy (EVmIM). EVs were endogenously loaded with mRNA (M) and protein (P) encoding an antigen (X) for the design of EVX-M+P to induce rapid and robust adaptive immune response and protection from future exposure. As a proof of concept, spike protein of SARS-CoV-2 and human ovalbumin (OVA) were used successfully as antigens for vaccines against a viral disease and melanoma, respectively. Next, EVs were endogenously loaded to harbor multiple surface immunomodulatory proteins of CD80, 4-1BBL, CD40L, CD2, and CD32 to generate EVmIM, which could induce APCs and T cells activation simultaneously to strengthen the antigen presentation and immune response against cancer progression, validated in orthotopic melanoma mouse model. The simplicity of EVs modification and cargo loading, and successful testing of the EVX-M+P and EVmIM platforms, offer new methodologies that can streamline the development of a new class of vaccines and immunotherapies. Collectively, my thesis research opens novel vaccination and cancer immunotherapy strategies that can be developed for human testing.Item Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2(Springer Nature, 2022) El-Shennawy, Lamiaa; Hoffmann, Andrew D.; Dashzeveg, Nurmaa Khund; McAndrews, Kathleen M.; Mehl, Paul J.; Cornish, Daphne; Yu, Zihao; Tokars, Valerie L.; Nicolaescu, Vlad; Tomatsidou, Anastasia; Mao, Chengsheng; Felicelli, Christopher J.; Tsai, Chia-Feng; Ostiguin, Carolina; Jia, Yuzhi; Li, Lin; Furlong, Kevin; Wysocki, Jan; Luo, Xin; Ruivo, Carolina F.; Batlle, Daniel; Hope, Thomas J.; Shen, Yang; Chae, Young Kwang; Zhang, Hui; LeBleu, Valerie S.; Shi, Tujin; Swaminathan, Suchitra; Luo, Yuan; Missiakas, Dominique; Randall, Glenn C.; Demonbreun, Alexis R.; Ison, Michael G.; Kalluri, Raghu; Fang, Deyu; Liu, Huiping; BioengineeringThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of the coronavirus induced disease 2019 (COVID-19) with evolving variants of concern. It remains urgent to identify novel approaches against broad strains of SARS-CoV-2, which infect host cells via the entry receptor angiotensin-converting enzyme 2 (ACE2). Herein, we report an increase in circulating extracellular vesicles (EVs) that express ACE2 (evACE2) in plasma of COVID-19 patients, which levels are associated with severe pathogenesis. Importantly, evACE2 isolated from human plasma or cells neutralizes SARS-CoV-2 infection by competing with cellular ACE2. Compared to vesicle-free recombinant human ACE2 (rhACE2), evACE2 shows a 135-fold higher potency in blocking the binding of the viral spike protein RBD, and a 60- to 80-fold higher efficacy in preventing infections by both pseudotyped and authentic SARS-CoV-2. Consistently, evACE2 protects the hACE2 transgenic mice from SARS-CoV-2-induced lung injury and mortality. Furthermore, evACE2 inhibits the infection of SARS-CoV-2 variants (α, β, and δ) with equal or higher potency than for the wildtype strain, supporting a broad-spectrum antiviral mechanism of evACE2 for therapeutic development to block the infection of existing and future coronaviruses that use the ACE2 receptor.Item Enacting national social distancing policies corresponds with dramatic reduction in COVID19 infection rates(Public Library of Science, 2020) McGrail, Daniel J.; Dai, Jianli; McAndrews, Kathleen M.; Kalluri, RaghuThe outbreak the SARS-CoV-2 (CoV-2) virus has resulted in over 6.5 million cases of COVID19, greatly stressing global healthcare infrastructure. Lacking medical prophylactic measures to combat disease spread, many nations have adopted social distancing policies in order to mitigate transmission of CoV-2. While mathematical models have suggested the efficacy of social distancing to curb the spread of CoV-2, there is a lack of systematic studies to quantify the real-world efficacy of these approaches. Here, we first demonstrate that implementation of social distancing policies in US states corresponded with a reduction in COVID19 spread rates, and that the reduction in spread rate is proportional to the average change in mobility. We validate this observation on a worldwide scale by analyzing COVID19 spread rate in 134 nations with varying social distancing policies. Globally, we find that social distancing policies significantly reduced the COVID19 spread rate, with resulting in an estimated 65% reduction (95% CI = 39–80%) in new COVID19 cases over a two week time period. These data suggest that social distancing policies may be a powerful tool to prevent spread of COVID19 in real-world scenarios.Item Exosomes modified with anti-MEK1 siRNA lead to an effective silencing of triple negative breast cancer cells(Elsevier, 2023) Ferreira, Débora; Santos-Pereira, Cátia; Costa, Marta; Afonso, Julieta; Yang, Sujuan; Hensel, Janine; McAndrews, Kathleen M.; Longatto-Filho, Adhemar; Fernandes, Rui; Melo, Joana B.; Baltazar, Fátima; Moreira, João N.; Kalluri, Raghu; Rodrigues, Ligia R.; BioengineeringTriple negative breast cancer (TNBC) is a highly heterogenous disease not sensitive to endocrine or HER2 therapy and standardized treatment regimens are still missing. Therefore, development of novel TNBC treatment approaches is of utmost relevance. Herein, the potential of MAPK/ERK downregulation by RNAi-based therapeutics in a panel of mesenchymal stem-like TNBC cell lines was uncovered. Our data revealed that suppression of one of the central nodes of this signaling pathway, MEK1, affects proliferation, migration, and invasion of TNBC cells, that may be explained by the reversion of the epithelial-mesenchymal transition phenotype, which is facilitated by the MMP-2/MMP-9 downregulation. Moreover, an exosome-based system was successfully generated for the siRNA loading (iExoMEK1). Our data suggested absence of modification of the physical properties and general integrity of the iExoMEK1 comparatively to the unmodified counterparts. Such exosome-mediated downregulation of MEK1 led to a tumor regression accompanied by a decrease of angiogenesis using the chick chorioallantoic-membrane model. Our results highlight the potential of the targeting of MAPK/ERK cascade as a promising therapeutic approach against TNBC.Item Genetic reprogramming with stem cells regenerates glomerular epithelial podocytes in Alport syndrome(Embo Press, 2024) LeBleu, Valerie S.; Kanasaki, Keizo; Lovisa, Sara; Alge, Joseph L.; Kim, Jiha; Chen, Yang; Teng, Yingqi; Gerami-Naini, Behzad; Sugimoto, Hikaru; Kato, Noritoshi; Revuelta, Ignacio; Grau, Nicole; Sleeman, Jonathan P.; Taduri, Gangadhar; Kizu, Akane; Rafii, Shahin; Hochedlinger, Konrad; Quaggin, Susan E.; Kalluri, Raghu; BioengineeringGlomerular filtration relies on the type IV collagen (ColIV) network of the glomerular basement membrane, namely, in the triple helical molecules containing the α3, α4, and α5 chains of ColIV. Loss of function mutations in the genes encoding these chains (Col4a3, Col4a4, and Col4a5) is associated with the loss of renal function observed in Alport syndrome (AS). Precise understanding of the cellular basis for the patho-mechanism remains unknown and a specific therapy for this disease does not currently exist. Here, we generated a novel allele for the conditional deletion of Col4a3 in different glomerular cell types in mice. We found that podocytes specifically generate α3 chains in the developing glomerular basement membrane, and that its absence is sufficient to impair glomerular filtration as seen in AS. Next, we show that horizontal gene transfer, enhanced by TGFβ1 and using allogenic bone marrow–derived mesenchymal stem cells and induced pluripotent stem cells, rescues Col4a3 expression and revive kidney function in Col4a3-deficient AS mice. Our proof-of-concept study supports that horizontal gene transfer such as cell fusion enables cell-based therapy in Alport syndrome.Item Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity(American Society for Clinical Investigation, 2020) McAndrews, Kathleen M.; Dowlatshahi, Dara P.; Dai, Jianli; Becker, Lisa M.; Hensel, Janine; Snowden, Laura M.; Leveille, Jennifer M.; Brunner, Michael R.; Holden, Kylie W.; Hopkins, Nikolas S.; Harris, Alexandria M.; Kumpati, Jerusha; Whitt, Michael A.; Lee, J. Jack; Ostrosky-Zeichner, Luis L.; Papanna, Ramesha; LeBleu, Valerie S.; Allison, James P.; Kalluri, RaghuEvaluation of potential immunity against the novel severe acute respiratory syndrome (SARS) coronavirus that emerged in 2019 (SARS-CoV-2) is essential for health, as well as social and economic recovery. Generation of antibody response to SARS-CoV-2 (seroconversion) may inform on acquired immunity from prior exposure, and antibodies against the SARS-CoV-2 spike protein receptor binding domain (S-RBD) are speculated to neutralize virus infection. Some serology assays rely solely on SARS-CoV-2 nucleocapsid protein (N-protein) as the antibody detection antigen; however, whether such immune responses correlate with S-RBD response and COVID-19 immunity remains unknown. Here, we generated a quantitative serological ELISA using recombinant S-RBD and N-protein for the detection of circulating antibodies in 138 serial serum samples from 30 reverse transcription PCR–confirmed, SARS-CoV-2–hospitalized patients, as well as 464 healthy and non–COVID-19 serum samples that were collected between June 2017 and June 2020. Quantitative detection of IgG antibodies against the 2 different viral proteins showed a moderate correlation. Antibodies against N-protein were detected at a rate of 3.6% in healthy and non–COVID-19 sera collected during the pandemic in 2020, whereas 1.9% of these sera were positive for S-RBD. Approximately 86% of individuals positive for S-RBD–binding antibodies exhibited neutralizing capacity, but only 74% of N-protein–positive individuals exhibited neutralizing capacity. Collectively, our studies show that detection of N-protein–binding antibodies does not always correlate with presence of S-RBD–neutralizing antibodies and caution against the extensive use of N-protein–based serology testing for determination of potential COVID-19 immunity.Item High throughput and rapid isolation of extracellular vesicles and exosomes with purity using size exclusion liquid chromatography(Elsevier, 2024) Kapoor, Kshipra S.; Harris, Kristen; Arian, Kent A.; Ma, Lihua; Schueng Zancanela, Beatriz; Church, Kaira A.; McAndrews, Kathleen M.; Kalluri, Raghu; BioengineeringExtracellular vesicles (EVs) have emerged as potential biomarkers for diagnosing a range of diseases without invasive procedures. Extracellular vesicles also offer advantages compared to synthetic vesicles for delivery of various drugs; however, limitations in segregating EVs from other particles and soluble proteins have led to inconsistent EV retrieval rates with low levels of purity. Here, we report a new high-yield (88.47 %) and rapid (<20 min) EV isolation method termed size exclusion – fast protein liquid chromatography (SE-FPLC). We show SE-FPLC can effectively isolate EVs from multiple sources including EVs derived from human and mouse cells and serum samples. The results indicate that SE-FPLC can successfully remove highly abundant protein contaminants such as albumin and lipoprotein complexes, which can represent a major hurdle in large scale isolation of EVs. The high-yield nature of SE-FPLC allows for easy industrial scaling up of EV production for various clinical utilities. SE-FPLC also enables analysis of small volumes of blood for use in point-of-care diagnostics in the clinic. Collectively, SE-FPLC offers many advantages over current EV isolation methods and offers rapid clinical translation.Item Identification of unique α4 chain structure and conserved antiangiogenic activity of α3NC1 type IV collagen in zebrafish(Wiley, 2023) LeBleu, Valerie S.; Dai, Jianli; Tsutakawa, Susan; MacDonald, Brian A.; Alge, Joseph L.; Sund, Malin; Xie, Liang; Sugimoto, Hikaru; Tainer, John; Zon, Leonard I.; Kalluri, Raghu; BioengineeringBackground Type IV collagen is an abundant component of basement membranes in all multicellular species and is essential for the extracellular scaffold supporting tissue architecture and function. Lower organisms typically have two type IV collagen genes, encoding α1 and α2 chains, in contrast with the six genes in humans, encoding α1–α6 chains. The α chains assemble into trimeric protomers, the building blocks of the type IV collagen network. The detailed evolutionary conservation of type IV collagen network remains to be studied. Results We report on the molecular evolution of type IV collagen genes. The zebrafish α4 non-collagenous (NC1) domain, in contrast with its human ortholog, contains an additional cysteine residue and lacks the M93 and K211 residues involved in sulfilimine bond formation between adjacent protomers. This may alter α4 chain interactions with other α chains, as supported by temporal and anatomic expression patterns of collagen IV chains during the zebrafish development. Despite the divergence between zebrafish and human α3 NC1 domain (endogenous angiogenesis inhibitor, Tumstatin), the zebrafish α3 NC1 domain exhibits conserved antiangiogenic activity in human endothelial cells. Conclusions Our work supports type IV collagen is largely conserved between zebrafish and humans, with a possible difference involving the α4 chain.Item Microfluidic Electrophoresis Platform for Analysis of Pancreatic Cancer Exosomes(2019-11-07) Kapoor, Kshipra; Kalluri, Raghu; Kono, JunichiroPancreatic cancer (PaCa) has a dismal prognosis, due to extremely late diagnosis, aggressive local metastasis, and limited treatment options. Thus, there is an urgent need for early detection methods for PaCa to increase the likelihood of treatment success. Liquid biopsy (marker detection in biofluids) is the only feasible approach for early diagnosis, as the biopsy of pancreatic tissue is not practical in patients lacking overt symptoms, and only minimally invasive methods can be used for sample collection. Recent studies demonstrate the use of exosomes as a means to improve the sensitivity of liquid biopsies. Most of the ongoing work utilizes stand-alone immunoaffinity-based capture of disease-associated exosomes in body fluids. The drawback of this approach is the heterogeneity of the exosome population, as the protein marker(s) of interest are typically expressed only by specific tumor subtypes, and the use of a single marker may result in false-negative or false-positive results or limit the enrichment capacity. In this work, we develop the novel principle of exosome fractionation based on their charge. The feasibility of the approach is based on our findings, which show a strong correlation between the negative charge of the exosomes and PaCa aggressiveness. Further analysis identified the enhanced incorporation of phosphatidylserine and DNA fragments in the outer leaflet of exosomal membranes, as the molecular cause of the highly anionic nature of cancer-derived exosomes. The use of a label-free separation approach allows us to collect intact exosomes and enables a broader range of downstream molecular analyses, unavailable in the permanent modifications by the tagging molecules on exosomes incurred by immunoaffinity techniques. Hence allowing for a multiplexed detection of biophysical and biomolecular properties of exosomes. We have designed and developed a prototype microfluidic platform that uses electrophoresis principles to isolate highly anionic exosomes, which correspond to the cancer-derived exosome population in patient serum. Further development of such a device for the detection of highly anionic exosomes in patient sera, would yield a much-needed tool for early PaCa diagnosis.Item Morphological diversity of extracellular vesicles revealed by cryo-electron microscopy(ASEMV/AAEV, 10/1/2022) Kapoor, Kshipra S.; McAndrews, Kathleen M.; Biswal, Lisa S.; Kalluri, Raghu; BioengineeringIntroduction: Exosomes are extracellular vesicles 80-150 nm in diameter, containing proteins, mRNAs, microRNAs, and lipids reflecting the parent cell. While there has been an extensive characterization of the cargo incorporated in exosomes, a detailed morphological analysis of exosomes purified by various isolation techniques has not been performed. Objective: We aimed to determine the heterogeneity of exosomes morphology and if such morphological features are conserved across sample types. Methods: Our study used Cryogenic Electron Microscopy (Cryo-EM) to examine exosome size and morphology. Results: Our results revealed significant diversity in extracellular vesicle morphology independent of the isolation method, suggesting that morphological subpopulations of these vesicles exist. Based on their shape, our analysis classified exosomes into seven categories. In addition, we developed a semi-automatic image analysis framework to accurately characterize exosome attributes and distribution to facilitate reliable quantification of specific bio-nanoparticle features in Cryo-EM micrographs. Conclusions: Morphological features of exosomes inform their biophysical properties, which influence both biodistribution and biological activity in vivo. Our data demonstrating the innate morphological diversity of exosomes may have implications for improving the specificity and precision of exosome-delivered therapeutics. Conflict of interest: R.K. and MD Anderson Cancer Center hold patents in exosome biology and are stock equity holders in Codiak Biosciences Inc. R.K. is a consultant and a scientific advisor of Codiak Biosciences Inc.Item New technological advances in scalable manufacturing and biophysical characterization of extracellular vesicles in biomedicine(2024-04-16) Kapoor, Kshipra; Kalluri, Raghu; Kono, JunichiroExtracellular Vesicles (EVs) have emerged as important mediators of intercellular communication that package and disseminate biochemical signals. This newly recognized mode of communication between the cells has brought unprecedented therapeutic and diagnostic opportunities making them attractive nanocarriers for clinical and industrial translation. As the EV industry rapidly grows, there is a rising demand for strategies that facilitate EV manufacturing. One of the most vexing issues in the field is a method of EV isolation that can offer reliability, purity, speed, and reproducibility and meet the stringent manufacturing standards of the pharmaceutical industry. To overcome this challenge, in the first part of my thesis, I propose a new highyield and rapid (<20 min) real-time EV isolation method called Size Exclusion – Fast Performance Liquid Chromatography (SE-FPLC). We show that our method can effectively isolate EVs from multiple sources, including EVs derived from human and mouse cells and biofluids. The results indicate that our SE-FPLC platform can successfully remove highly abundant protein contaminants, such as albumin and lipoprotein complexes, which currently represent a significant hurdle in the largescale isolation of EVs for clinical translation. Additionally, the high-yield nature of SE-FPLC allows for easy industrial upscaling of EV production for various clinical utilities. Moreover, SE-FPLC enables analysis of very small volumes of blood for use in point-of-care diagnostics in the clinic. Collectively, our platform offers many advantages over current EV isolation methods and offers rapid clinical utility potential. Once the EVs are isolated, it is imperative to perform EV physicochemical characterization as particle shape and particle charge is pivotal in immune cell interaction. Bulk ensemble methods quantify EV composition but mask heterogeneity. Studying single-vesicle heterogeneity is vital, especially given their emerging role as therapeutic cargos. In the second part of my thesis, I developed a label-free method: to image, perform high-quality biological segmentation using a custom pre-trained neural network model, and quantify and classify single EVs purified from a diverse set of samples. Evaluating the heterogeneity of EVs is crucial for unraveling their complex actions and biodistribution. We identified consistent architectural heterogeneity of EVs using cryogenic transmission electron microscopy (cryo-TEM). Imaging EVs isolated using different methodologies from distinct sources such as cancer cells, normal cells, and body fluids, we identify a structural atlas of their dominantly consistent shapes. We identify EV architectural attributes by utilizing a segmentation neural network model. In total, 7,600 individual EVs were imaged and quantified by our computational pipeline. Across all 7,600 independent EVs, the average eccentricity was 0.5366, and the average equivalent diameter was 132.43 nm. The architectural heterogeneity was consistent across all sources of EVs, independent of purification techniques, and compromised of single spherical (S. Spherical), rod-like or tubular, and double shapes. This openly accessible data and computation toolkit will serve as a reference foundation for high-resolution EV images and offer insights into potential biological impact.Item A novel machine learning algorithm selects proteome signature to specifically identify cancer exosomes(eLife Sciences Publications Ltd., 2024) Li, Bingrui; Kugeratski, Fernanda G; Kalluri, Raghu; BioengineeringNon-invasive early cancer diagnosis remains challenging due to the low sensitivity and specificity of current diagnostic approaches. Exosomes are membrane-bound nanovesicles secreted by all cells that contain DNA, RNA, and proteins that are representative of the parent cells. This property, along with the abundance of exosomes in biological fluids makes them compelling candidates as biomarkers. However, a rapid and flexible exosome-based diagnostic method to distinguish human cancers across cancer types in diverse biological fluids is yet to be defined. Here, we describe a novel machine learning-based computational method to distinguish cancers using a panel of proteins associated with exosomes. Employing datasets of exosome proteins from human cell lines, tissue, plasma, serum, and urine samples from a variety of cancers, we identify Clathrin Heavy Chain (CLTC), Ezrin, (EZR), Talin-1 (TLN1), Adenylyl cyclase-associated protein 1 (CAP1), and Moesin (MSN) as highly abundant universal biomarkers for exosomes and define three panels of pan-cancer exosome proteins that distinguish cancer exosomes from other exosomes and aid in classifying cancer subtypes employing random forest models. All the models using proteins from plasma, serum, or urine-derived exosomes yield AUROC scores higher than 0.91 and demonstrate superior performance compared to Support Vector Machine, K Nearest Neighbor Classifier and Gaussian Naive Bayes. This study provides a reliable protein biomarker signature associated with cancer exosomes with scalable machine learning capability for a sensitive and specific non-invasive method of cancer diagnosis.Item Protection against SARS-CoV-2 by BCG vaccination is not supported by epidemiological analyses(Springer Nature, 2020) Hensel, Janine; McAndrews, Kathleen M.; McGrail, Daniel J.; Dowlatshahi, Dara P.; LeBleu, Valerie S.; Kalluri, RaghuThe Bacillus Calmette-Guerin (BCG) vaccine provides protection against tuberculosis (TB), and is thought to provide protection against non-TB infectious diseases. BCG vaccination has recently been proposed as a strategy to prevent infection with SARS-CoV-2 (CoV-2) to combat the COVID-19 outbreak, supported by its potential to boost innate immunity and initial epidemiological analyses which observed reduced severity of COVID-19 in countries with universal BCG vaccination policies. Seventeen clinical trials are currently registered to inform on the benefits of BCG vaccinations upon exposure to CoV-2. Numerous epidemiological analyses showed a correlation between incidence of COVID-19 and BCG vaccination policies. These studies were not systematically corrected for confounding variables. We observed that after correction for confounding variables, most notably testing rates, there was no association between BCG vaccination policy and COVD-19 spread rate or percent mortality. Moreover, we found variables describing co-morbidities, including cardiovascular death rate and smoking prevalence, were significantly associated COVID-19 spread rate and percent mortality, respectively. While reporting biases may confound our observations, our epidemiological findings do not provide evidence to correlate overall BCG vaccination policy with the spread of CoV-2 and its associated mortality.Item Type-I collagen produced by distinct fibroblast lineages reveals specific function during embryogenesis and Osteogenesis Imperfecta(Springer Nature, 2021) Chen, Yang; Yang, Sujuan; Lovisa, Sara; Ambrose, Catherine G.; McAndrews, Kathleen M.; Sugimoto, Hikaru; Kalluri, Raghu; BioengineeringType I collagen (Col1) is the most abundant protein in mammals. Col1 contributes to 90% of the total organic component of bone matrix. However, the precise cellular origin and functional contribution of Col1 in embryogenesis and bone formation remain unknown. Single-cell RNA-sequencing analysis identifies Fap+ cells and Fsp1+ cells as the major contributors of Col1 in the bone. We generate transgenic mouse models to genetically delete Col1 in various cell lineages. Complete, whole-body Col1 deletion leads to failed gastrulation and early embryonic lethality. Specific Col1 deletion in Fap+ cells causes severe skeletal defects, with hemorrhage, edema, and prenatal lethality. Specific Col1 deletion in Fsp1+ cells results in Osteogenesis Imperfecta-like phenotypes in adult mice, with spontaneous fractures and compromised bone healing. This study demonstrates specific contributions of mesenchymal cell lineages to Col1 production in organogenesis, skeletal development, and bone formation/repair, with potential insights into cell-based therapy for patients with Osteogenesis Imperfecta.Item Unique somatic variants in DNA from urine exosomes of individuals with bladder cancer(Elsevier, 2021) Zhou, Xunian; Kurywchak, Paul; Wolf-Dennen, Kerri; Che, Sara P. Y.; Sulakhe, Dinanath; D’Souza, Mark; Xie, Bingqing; Maltsev, Natalia; Gilliam, T. Conrad; Wu, Chia-Chin; McAndrews, Kathleen M.; LeBleu, Valerie S.; McConkey, David J.; Volpert, Olga V.; Pretzsch, Shanna M.; Czerniak, Bogdan A.; Dinney, Colin P.; Kalluri, Raghu; BioengineeringBladder cancer (BC), a heterogeneous disease characterized by high recurrence rates, is diagnosed and monitored by cystoscopy. Accurate clinical staging based on biopsy remains a challenge, and additional, objective diagnostic tools are needed urgently. We used exosomal DNA (exoDNA) as an analyte to examine cancer-associated mutations and compared the diagnostic utility of exoDNA from urine and serum of individuals with BC. In contrast to urine exosomes from healthy individuals, urine exosomes from individuals with BC contained significant amounts of DNA. Whole-exome sequencing of DNA from matched urine and serum exosomes, bladder tumors, and normal tissue (peripheral blood mononuclear cells) identified exonic and 3′ UTR variants in frequently mutated genes in BC, detectable in urine exoDNA and matched tumor samples. Further analyses identified somatic variants in driver genes, unique to urine exoDNA, possibly because of the inherent intra-tumoral heterogeneity of BC, which is not fully represented in random small biopsies. Multiple variants were also found in untranslated portions of the genome, such as microRNA (miRNA)-binding regions of the KRAS gene. Gene network analyses revealed that exoDNA is associated with cancer, inflammation, and immunity in BC exosomes. Our findings show utility of exoDNA as an objective, non-invasive strategy to identify novel biomarkers and targets for BC.