Research Lines
Tumor Exosome Biology
Tumor-derived exosomes are increasingly recognized as important players in cell-cell communication and have been implicated in cancer initiation and stimulate invasive behavior of cancer cells leading to metastasis. However, insight into the regulatory control of exosome release dynamics hampers direct demonstration on their actions in vitro and in vivo. We developed methods that can visualize exosome release from living cancer cells. Live total internal fluorescence (TIRF)-microscopy can demonstrate that tumor microenvironment (TME) components released by tumor stroma cells activate signaling pathways that in turn seem to promote fusion of intracellular compartments with the plasma membrane resulting in exosomes release. This knowledge will be exploited to discover novel drug targets.
Tumor Exosome Biology
Tumor-derived exosomes are increasingly recognized as important players in cell-cell communication and have been implicated in cancer initiation and stimulate invasive behavior of cancer cells leading to metastasis. However, insight into the regulatory control of exosome release dynamics hampers direct demonstration on their actions in vitro and in vivo. We developed methods that can visualize exosome release from living cancer cells. Live total internal fluorescence (TIRF)-microscopy can demonstrate that tumor microenvironment (TME) components released by tumor stroma cells activate signaling pathways that in turn seem to promote fusion of intracellular compartments with the plasma membrane resulting in exosomes release. This knowledge will be exploited to discover novel drug targets.
Figure: Projection of exosome release events (yellow spots) in a cervical cancer cell (blue) over a time-course of 3 minutes.
Extracellular Vesicle RNA biology and diagnostics
The genetic profiling of patient material has yielded reliable prognostic biomarkers that are paramount for early detection, diagnosis and assessment of effective therapy. One drawback is that patient tissue is not always available or accessible without conducting timely and painful procedures. The presence of small genetic elements called ‘microRNAs’ in extracellular vesicles (EV) or stably bound to proteins in patient biofluids opens unique possibilities for using such circulating RNAs as easily accessible genetic biomarkers. Our laboratory uses multiple strategies for development of ‘liquid biopsy’ tests that should enable non-invasive diagnosis of cancer in the future, for instance by a simple blood test. We are using similar strategies in severe autoimmune patients with chronic inflammation and discovered a link between EV physiology (exosomes), Epstein Barr virus infection and antiviral immunity. For these studies we use the latest high-throughput next-generation sequencing techniques to unravel the complete small RNA landscape of exosomes. We have found fundamental differences between small RNAs in tissues/cell when compared to exosomes, pointing to a function outside the cell in which they were produced. Currently we investigate the possibility that small distinctions in extracellular small RNA content maybe applicable for non-invasive disease monitoring, primarily by isolation of exosomes from patient blood and urine (van Eijndhoven et al., 2016).
Development of novel Next Generation Sequencing methods
Secreted microRNAs (miRNAs) are proposed as powerful disease indicators and when associated with extracellular vesicles (EVs), have an active role in organ crosstalk. Because the majority of miRNAs are broadly expressed across cell-types and tissues, a single miRNA is rarely informative for a disease condition. Recent advances show that non-templated nucleotide additions (isomiRs) change miRNA gene-regulatory functions and incorporating this additional layer of complexity increases diagnostic accuracy of miRNAs. Small RNA sequencing is the only method that gains comprehensive insight into the diversity and relative quantities of miRNAs and their subspecies. We show here that, despite improvements, current small RNAseq methods remain imprecise. The accuracy of cell-free miRNA detection is particularly low due to low input amounts which may underlie difficulties in demonstrating clinical utility. The method we developed (isoSeq) makes use of 5N-randomized 3’ and 5’ adapters and unique molecular identifier (UMI) dramatically reducing bias in detection. With custom-designed synthetic isomiR spike-ins and cell-lines lacking terminal uridylases (TUT4 and TUT7), we validated that isoSeq provides robust, quantitative and qualitative information on miRNA complexity from circulating EVs, an increasingly used liquid biosource. IsoSeq will improve our understanding of miRNA biology and their adaption as minimally-invasive disease indicators.
Key publications
Towards IVDR-compliance by implementing quality control steps in a quantitative extracellular vesicle-miRNA liquid biopsy assay for response monitoring in patients with classic Hodgkin lymphoma.
Drees EEE, Groenewegen NJ, Verkuijlen SAWM, van Eijndhoven MAJ, Ramaker J, Veenstra P, Hussain M, Groothuis-Oudshoorn CGM, de Jong D, Zijlstra JM, de Rooij J, Pegtel DM. J Extracell Biol. 2024 Jun 28;3(7):e164. doi: 10.1002/jex2.164. eCollection 2024 Jul. PMID: 38947877
IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolution
Monique A J van Eijndhoven 1, Chantal Scheepbouwer 2, Ernesto Aparicio-Puerta 3, Michael Hackenberg 3, D Michiel Pegtel 4
STAR Protoc. 2023 Dec 15;4(4):102645. doi: 10.1016/j.xpro.2023.102645. Epub 2023 Oct 19.
Reassessment of miRNA variant (isomiRs) composition by small RNA sequencing
Gómez-Martín C, Aparicio-Puerta E, van Eijndhoven MAJ, Medina JM, Hackenberg M, Pegtel DM. Cell Reports Methods. 2023 May
Real-time imaging of multivesicular body-plasma membrane fusion to quantify exosome release from single cells.
Bebelman MP, Bun P, Huveneers S, van Niel G, Verweij FJ, Pegtel DM. Nat Protoc. 2020 Jan;15(1):102-121. doi: 10.1038/s41596-019-0245-4. Epub 2019 Dec 13. PMID: 31836866
Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling
Verweij FJ, Bebelman MP, Jimenez CR, Garcia-Vallejo JJ, Janssen H, Neefjes J, Knol JC, de Goeij-de Haas R, Piersma SR, Baglio SR, Verhage M, Middeldorp JM, Zomer A, van Rheenen J, Coppolino MG, Hurbain I, Raposo G, Smit MJ, Toonen RFG, van Niel G, Pegtel DM. J Cell Biol. 2018 Jan 16.
Sensing of latent EBV infection through exosomal transfer of 5'pppRNA.
Baglio SR, van Eijndhoven MA, Koppers-Lalic D, Berenguer J, Lougheed SM, Gibbs S, Léveillé N, Rinkel RN, Hopmans ES, Swaminathan S, Verkuijlen SA, Scheffer GL, van Kuppeveld FJ, de Gruijl TD, Bultink IE, Jordanova ES, Hackenberg M, Piersma SR, Knol JC, Voskuyl AE, Wurdinger T, Jiménez CR, Middeldorp JM, Pegtel DM. Proc Natl Acad Sci U S A. 2016 Feb 2;113(5):E587-96. doi: 10.1073/pnas.1518130113. Epub 2016 Jan 14. PMID: 26768848
Nontemplated nucleotide additions distinguish the small RNA composition in cells from exosomes
Koppers-Lalic D, Hackenberg M, Bijnsdorp IV, van Eijndhoven MAJ, Sadek P, Sie D, Zini N, Middeldorp JM, Ylstra B, de Menezes RX, Wurdinger T, Meijer GA, and Pegtel DM. Cell Rep. 2014;8(6):1649–58.
LMP1 association with CD63 in endosomes and secretion via exosomes limits constitutive NF-κB activation
Verweij FJ, van Eijndhoven MA, Hopmans ES, Vendrig T, Wurdinger T, Cahir-McFarland E, Kieff E, Geerts D, van der Kant R, Neefjes J, Middeldorp JM, Pegtel DM. EMBO Journal 2011; 30: 2115-2129
Functional delivery of viral miRNAs via exosomes
Pegtel DM, Cosmopoulos K, Thorley-Lawson DA, van Eijndhoven MA, Hopmans ES, et al. Proc Natl Acad Sci USA. 2010;107:6328-6333.
The genetic profiling of patient material has yielded reliable prognostic biomarkers that are paramount for early detection, diagnosis and assessment of effective therapy. One drawback is that patient tissue is not always available or accessible without conducting timely and painful procedures. The presence of small genetic elements called ‘microRNAs’ in extracellular vesicles (EV) or stably bound to proteins in patient biofluids opens unique possibilities for using such circulating RNAs as easily accessible genetic biomarkers. Our laboratory uses multiple strategies for development of ‘liquid biopsy’ tests that should enable non-invasive diagnosis of cancer in the future, for instance by a simple blood test. We are using similar strategies in severe autoimmune patients with chronic inflammation and discovered a link between EV physiology (exosomes), Epstein Barr virus infection and antiviral immunity. For these studies we use the latest high-throughput next-generation sequencing techniques to unravel the complete small RNA landscape of exosomes. We have found fundamental differences between small RNAs in tissues/cell when compared to exosomes, pointing to a function outside the cell in which they were produced. Currently we investigate the possibility that small distinctions in extracellular small RNA content maybe applicable for non-invasive disease monitoring, primarily by isolation of exosomes from patient blood and urine (van Eijndhoven et al., 2016).
Development of novel Next Generation Sequencing methods
Secreted microRNAs (miRNAs) are proposed as powerful disease indicators and when associated with extracellular vesicles (EVs), have an active role in organ crosstalk. Because the majority of miRNAs are broadly expressed across cell-types and tissues, a single miRNA is rarely informative for a disease condition. Recent advances show that non-templated nucleotide additions (isomiRs) change miRNA gene-regulatory functions and incorporating this additional layer of complexity increases diagnostic accuracy of miRNAs. Small RNA sequencing is the only method that gains comprehensive insight into the diversity and relative quantities of miRNAs and their subspecies. We show here that, despite improvements, current small RNAseq methods remain imprecise. The accuracy of cell-free miRNA detection is particularly low due to low input amounts which may underlie difficulties in demonstrating clinical utility. The method we developed (isoSeq) makes use of 5N-randomized 3’ and 5’ adapters and unique molecular identifier (UMI) dramatically reducing bias in detection. With custom-designed synthetic isomiR spike-ins and cell-lines lacking terminal uridylases (TUT4 and TUT7), we validated that isoSeq provides robust, quantitative and qualitative information on miRNA complexity from circulating EVs, an increasingly used liquid biosource. IsoSeq will improve our understanding of miRNA biology and their adaption as minimally-invasive disease indicators.
Key publications
Towards IVDR-compliance by implementing quality control steps in a quantitative extracellular vesicle-miRNA liquid biopsy assay for response monitoring in patients with classic Hodgkin lymphoma.
Drees EEE, Groenewegen NJ, Verkuijlen SAWM, van Eijndhoven MAJ, Ramaker J, Veenstra P, Hussain M, Groothuis-Oudshoorn CGM, de Jong D, Zijlstra JM, de Rooij J, Pegtel DM. J Extracell Biol. 2024 Jun 28;3(7):e164. doi: 10.1002/jex2.164. eCollection 2024 Jul. PMID: 38947877
IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolution
Monique A J van Eijndhoven 1, Chantal Scheepbouwer 2, Ernesto Aparicio-Puerta 3, Michael Hackenberg 3, D Michiel Pegtel 4
STAR Protoc. 2023 Dec 15;4(4):102645. doi: 10.1016/j.xpro.2023.102645. Epub 2023 Oct 19.
Reassessment of miRNA variant (isomiRs) composition by small RNA sequencing
Gómez-Martín C, Aparicio-Puerta E, van Eijndhoven MAJ, Medina JM, Hackenberg M, Pegtel DM. Cell Reports Methods. 2023 May
Real-time imaging of multivesicular body-plasma membrane fusion to quantify exosome release from single cells.
Bebelman MP, Bun P, Huveneers S, van Niel G, Verweij FJ, Pegtel DM. Nat Protoc. 2020 Jan;15(1):102-121. doi: 10.1038/s41596-019-0245-4. Epub 2019 Dec 13. PMID: 31836866
Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling
Verweij FJ, Bebelman MP, Jimenez CR, Garcia-Vallejo JJ, Janssen H, Neefjes J, Knol JC, de Goeij-de Haas R, Piersma SR, Baglio SR, Verhage M, Middeldorp JM, Zomer A, van Rheenen J, Coppolino MG, Hurbain I, Raposo G, Smit MJ, Toonen RFG, van Niel G, Pegtel DM. J Cell Biol. 2018 Jan 16.
Sensing of latent EBV infection through exosomal transfer of 5'pppRNA.
Baglio SR, van Eijndhoven MA, Koppers-Lalic D, Berenguer J, Lougheed SM, Gibbs S, Léveillé N, Rinkel RN, Hopmans ES, Swaminathan S, Verkuijlen SA, Scheffer GL, van Kuppeveld FJ, de Gruijl TD, Bultink IE, Jordanova ES, Hackenberg M, Piersma SR, Knol JC, Voskuyl AE, Wurdinger T, Jiménez CR, Middeldorp JM, Pegtel DM. Proc Natl Acad Sci U S A. 2016 Feb 2;113(5):E587-96. doi: 10.1073/pnas.1518130113. Epub 2016 Jan 14. PMID: 26768848
Nontemplated nucleotide additions distinguish the small RNA composition in cells from exosomes
Koppers-Lalic D, Hackenberg M, Bijnsdorp IV, van Eijndhoven MAJ, Sadek P, Sie D, Zini N, Middeldorp JM, Ylstra B, de Menezes RX, Wurdinger T, Meijer GA, and Pegtel DM. Cell Rep. 2014;8(6):1649–58.
LMP1 association with CD63 in endosomes and secretion via exosomes limits constitutive NF-κB activation
Verweij FJ, van Eijndhoven MA, Hopmans ES, Vendrig T, Wurdinger T, Cahir-McFarland E, Kieff E, Geerts D, van der Kant R, Neefjes J, Middeldorp JM, Pegtel DM. EMBO Journal 2011; 30: 2115-2129
Functional delivery of viral miRNAs via exosomes
Pegtel DM, Cosmopoulos K, Thorley-Lawson DA, van Eijndhoven MA, Hopmans ES, et al. Proc Natl Acad Sci USA. 2010;107:6328-6333.