Kasper Johnsen - Academia.edu (original) (raw)

Papers by Kasper Johnsen

The blood-brain barrier (BBB), built by brain endothelial cells (BECs), is impermeable to biologi... more The blood-brain barrier (BBB), built by brain endothelial cells (BECs), is impermeable to biologics. Liposomes and other nanoparticles are good candidates for delivery of biologics across the BECs, as they can encapsulate numerous molecules of interest in an omnipotent manner. The liposomes need attachment of a targeting molecule, as BECs unfortunately are virtually incapable of uptake of non-targeted liposomes from the circulation. Experiments of independent research groups have qualified antibodies targeting the transferrin receptor as superior for targeted delivery of nanoparticles to BECs. Functionalization of nanoparticles via conjugation with anti-transferrin receptor antibodies leads to nanoparticle uptake by endothelial cells of both brain capillaries and post-capillary venules. Reducing the density of transferrin receptor-targeted antibodies conjugated to liposomes limits uptake in BECs. Opposing the transport of nanoparticles conjugated to high-affine anti-transferrin rece...

Drug Delivery to the Brain

Communications Biology, 2021

Precise methods for quantifying drug accumulation in brain tissue are currently very limited, cha... more Precise methods for quantifying drug accumulation in brain tissue are currently very limited, challenging the development of new therapeutics for brain disorders. Transcardial perfusion is instrumental for removing the intravascular fraction of an injected compound, thereby allowing for ex vivo assessment of extravasation into the brain. However, pathological remodeling of tissue microenvironment can affect the efficiency of transcardial perfusion, which has been largely overlooked. We show that, in contrast to healthy vasculature, transcardial perfusion cannot remove an injected compound from the tumor vasculature to a sufficient extent leading to considerable overestimation of compound extravasation. We demonstrate that 3D deep imaging of optically cleared tumor samples overcomes this limitation. We developed two machine learning-based semi-automated image analysis workflows, which provide detailed quantitative characterization of compound extravasation patterns as well as tumor a...

American Journal of Hematology, 2017

transferrin receptor antibody targeted gold nanoparticles DTU Orbit (12/11/2019) Modulating antib... more transferrin receptor antibody targeted gold nanoparticles DTU Orbit (12/11/2019) Modulating antibody affinity towards the transferrin receptor to increase brain uptake of anti-transferrin receptor antibody targeted gold nanoparticles Drug delivery to the brain is hampered by the presence of the blood-brain barrier (BBB) that under physiological conditions precludes entrance of most substances contained in the systemic circulation. Thus, this barrier must be overcome to deliver medicines into the brain parenchyma. The transferrin receptor is exclusively expressed on capillaries of the brain, which makes it an interesting target for transport of drugs towards the brain. However, the current evidence on the receptor movement in brain capillaries does not suggest transcytosis, and delivering medicines or nanoparticles using antibodies towards this receptor has largely been without success. We investigated the impact of antibody affinity on the transport of gold nanoparticles into the br...

Ultrasound in Medicine & Biology, 2021

The blood-brain barrier (BBB) is a major obstacle to treating several brain disorders. Focused ul... more The blood-brain barrier (BBB) is a major obstacle to treating several brain disorders. Focused ultrasound (FUS) in combination with intravascular microbubbles increases BBB permeability by opening tight junctions, creating endothelial cell openings, improving endocytosis and increasing transcytosis. Here we investigated whether combining FUS and microbubbles with transferrin receptor-targeting liposomes would result in enhanced delivery to the brain of post-natal rats compared with liposomes lacking the BBB-targeting moiety. For all animals, increased BBB permeability was observed after FUS treatment. A 40% increase in accumulation of transferrin receptor-targeting liposomes was observed in the FUS-treated hemisphere, whereas the isotype immunoglobulin G liposomes showed no increased accumulation. Confocal laser scanning microscopy of brain sections revealed that both types of liposomes were mainly observed in endothelial cells in the FUS-treated hemisphere. The results demonstrate that FUS and microbubble treatment combined with BBB-targeting liposomes could be a promising approach to enhance drug delivery to the brain.

The FEBS Journal, 2021

Brain homeostasis depends on the existence of the blood–brain barrier (BBB). Despite decades of r... more Brain homeostasis depends on the existence of the blood–brain barrier (BBB). Despite decades of research, the factors and signalling pathways for modulating and maintaining BBB integrity are not fully elucidated. Here, we characterise the expression and function of the multifunctional receptor, sortilin, in the cells of the BBB, in vivo and in vitro. We show that sortilin acts as an important regulatory protein of the BBB’s tightness. In rats lacking sortilin, the BBB was leaky, which correlated well with relocated distribution of the localisation of zonula occludens‐1, VE‐cadherin and β‐catenin junctional proteins. Furthermore, the absence of sortilin in brain endothelial cells resulted in decreased phosphorylation of Akt signalling protein and increased the level of phospho‐ERK1/2. As a putative result of MAPK/ERK pathway activity, the junctions between the brain endothelial cells were disintegrated and the integrity of the BBB became compromised. The identified barrier differences between wild‐type and Sort1−/− brain endothelial cells can pave the way for a better understanding of sortilin’s role in the healthy and diseased BBB.

Nature Communications, 2021

Effective treatments of neurodegenerative diseases require drugs to be actively transported acros... more Effective treatments of neurodegenerative diseases require drugs to be actively transported across the blood-brain barrier (BBB). However, nanoparticle drug carriers explored for this purpose show negligible brain uptake, and the lack of basic understanding of nanoparticle-BBB interactions underlies many translational failures. Here, using two-photon microscopy in mice, we characterize the receptor-mediated transcytosis of nanoparticles at all steps of delivery to the brain in vivo. We show that transferrin receptor-targeted liposome nanoparticles are sequestered by the endothelium at capillaries and venules, but not at arterioles. The nanoparticles move unobstructed within endothelium, but transcytosis-mediated brain entry occurs mainly at post-capillary venules, and is negligible in capillaries. The vascular location of nanoparticle brain entry corresponds to the presence of perivascular space, which facilitates nanoparticle movement after transcytosis. Thus, post-capillary venule...

Molecular Neurobiology, 2020

Glioblastoma (GBM) is the most frequent and devastating primary tumor of the central nervous syst... more Glioblastoma (GBM) is the most frequent and devastating primary tumor of the central nervous system with a median survival of 12 to 15 months after diagnosis. GBM is highly difficult to treat due to its delicate location, inter-and intra-tumoral heterogeneity, and high plasticity in response to treatment. In this study, we intracranially implanted primary GBM cells into mice which underwent conventional GBM treatments, including irradiation, temozolomide, and a combination. We obtained single cell suspensions through a combination of mechanical and enzymatic dissociation of brain tissue and investigated in detail the changes in GBM cells in response to conventional treatments in vivo using multi-color flow cytometry and cluster analysis. CD44 expression was elevated in all treatment groups, which was confirmed by subsequent immunohistochemistry. High CD44 expression was furthermore shown to correlate with poor prognosis of GBM and low-grade glioma (LGG) patients. Together, these results indicate a key role for CD44 in glioma pathogenesis.

SUMMARYTreatments of neurodegenerative diseases require biologic drugs to be actively transported... more SUMMARYTreatments of neurodegenerative diseases require biologic drugs to be actively transported across the blood-brain barrier (BBB). To answer outstanding questions regarding transport mechanisms, we determined how and where transcytosis occurs at the BBB. Using two-photon microscopy, we characterized the transport of therapeutic nanoparticles at all steps of delivery to the brain and at the nanoscale resolution in vivo. Transferrin receptor-targeted nanoparticles were taken up by endothelium at capillaries and venules, but not at arterioles. The nanoparticles moved unobstructed within endothelial cells, but transcytosis across the BBB occurred only at post-capillary venules, where endothelial and glial basement membranes form a perivascular space that can accommodate biologics. In comparison, transcytosis was absent in capillaries with closely apposed basement membranes. Thus, post-capillary venules, not capillaries, provide an entry point for transport of large molecules across...

Cancer and Metastasis Reviews, 2020

Cancer treatment remains a challenge due to a high level of intra- and intertumoral heterogeneity... more Cancer treatment remains a challenge due to a high level of intra- and intertumoral heterogeneity and the rapid development of chemoresistance. In the brain, this is further hampered by the blood-brain barrier that reduces passive diffusion of drugs to a minimum. Tumors grow invasively and form new blood vessels, also in brain tissue where remodeling of pre-existing vasculature is substantial. The cancer-associated vessels in the brain are considered leaky and thus could facilitate the transport of chemotherapeutic agents. Yet, brain tumors are extremely difficult to treat, and, in this review, we will address how different aspects of the vasculature in brain tumors contribute to this.

Progress in Neurobiology, 2019

Targeting of the transferrin receptor is one of the most efficient ways of obtaining brain drug d... more Targeting of the transferrin receptor is one of the most efficient ways of obtaining brain drug delivery.  Despite decades of development, very little clinical progression has been made, although few clinical trials are beginning to emerge.  New innovations in terms of modifications of the antibody binding mode has yielded impressive leaps forward in our understanding of the transport process.  Many parameters in the transferrin receptor-mediated transport across the blood-brain barrier are still poorly understood.  Increased understanding of factors like intracellular sorting and subsequent long-term brain retention of TfR-targeted drug delivery systems is needed to fulfill the ambition of clinical use.

Journal of Cell Communication and Signaling, 2019

Exosomes were first described as waste carriers implicated in reticulocyte maturation but has dur... more Exosomes were first described as waste carriers implicated in reticulocyte maturation but has during the past decade been associated with many other cellular functions. The biogenesis of exosomes has been extensively studied and several protein machineries have been identified to dictate their production and release. The newly discovered branches of the autophagy system implicate secretion of waste in endosomal-derived vesicles as is thought for exosome release. Many of the proteins that have been identified as responsible for the formation and release of these vesicles are the same as those identified in exosome biogenesis. In this Perspective, we discuss the possibility of exosomes being a part of the autophagy machinery and the consequences this could have on interpretation of exosome functions.

Journal of Controlled Release, 2019

Systematic review of targeted extracellular vesicles for drug delivery Considerations on methodol... more Systematic review of targeted extracellular vesicles for drug delivery Considerations on methodological and biological heterogeneity

Experimental Cell Research, 2019

A tumorsphere model of glioblastoma multiforme with intratumoral heterogeneity for quantitative a... more A tumorsphere model of glioblastoma multiforme with intratumoral heterogeneity for quantitative analysis of cellular migration and drug response

Journal of Controlled Release, 2019

Transport of the majority of therapeutic molecules to the brain is precluded by the presence of t... more Transport of the majority of therapeutic molecules to the brain is precluded by the presence of the blood-brain barrier (BBB) rendering efficient treatment of many neurological disorders impossible. This BBB, nonetheless, may be circumvented by targeting receptors and transport proteins expressed on the luminal surface of the brain capillary endothelial cells (BCECs). The transferrin receptor (TfR) has remained a popular target since its original description for this purpose, although clinical progression of TfR-targeted drug constructs or nanomedicines remains unsuccessful. One proposed issue pertaining to the use of TfR-targeting in nanomedicines is the efficient tuning of the ligand density on the nanoparticle surface. We studied the impact of TfR antibody density on the uptake and transport of nanoparticles into the brain, taking a parallel approach to investigate the impact on both antibody-functionalized gold nanoparticles (AuNPs) and cargo-loaded liposomes. We report that among three different low-range mean ligand densities (0.15, 0.3, and 0.6 * 10 3 antibodies/μm 2), the highest density yielded the highest ability towards both targeting of the BCECs and subsequent transport across the BBB in vivo, and in vitro using primary cultures of the murine BBB. We also find that TfR-targeting on liposomes in the mouse may induce severe adverse effects after intravenous administration.

Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2018

Circulating biomarkers have a great potential in diagnosing cancer diseases at early stages, wher... more Circulating biomarkers have a great potential in diagnosing cancer diseases at early stages, where curative treatment is a realistic possibility. In the recent years, using extracellular vesicles (EVs) derived from blood as biomarkers has gained widespread popularity, mainly because they are thought to be easy to isolate and carry a vast variety of biological cargos that can be analyzed for biomarker purposes. However, our current knowledge on the plasma EV concentration in normophysiological states is sparse. Here, we provide the very first mean estimate of the plasma EV concentration based on values obtained from a thorough literature review. The different estimates obtained from the literature are correlated to the isolation techniques used to obtain them, illustrating how some methodologies may over-or underestimate the plasma EV concentration. We also show that the estimated plasma EV concentration (approximately 10 10 EVs per mL) defines EVs as a minority population compared to other colloidal particles of the systemic circulation, namely the lipoproteins, which are known contaminants in EV isolates and carry biomarker molecules themselves. Lastly, we introduce the possibility of regarding EVs and lipoproteins as a continuum of lipid-containing particles to which biomarker molecules can be associated. Using such a holistic approach, increased strength of plasma-derived cancer biomarkers may soon be revealed.

The blood-brain barrier (BBB), built by brain endothelial cells (BECs), is impermeable to biologi... more The blood-brain barrier (BBB), built by brain endothelial cells (BECs), is impermeable to biologics. Liposomes and other nanoparticles are good candidates for delivery of biologics across the BECs, as they can encapsulate numerous molecules of interest in an omnipotent manner. The liposomes need attachment of a targeting molecule, as BECs unfortunately are virtually incapable of uptake of non-targeted liposomes from the circulation. Experiments of independent research groups have qualified antibodies targeting the transferrin receptor as superior for targeted delivery of nanoparticles to BECs. Functionalization of nanoparticles via conjugation with anti-transferrin receptor antibodies leads to nanoparticle uptake by endothelial cells of both brain capillaries and post-capillary venules. Reducing the density of transferrin receptor-targeted antibodies conjugated to liposomes limits uptake in BECs. Opposing the transport of nanoparticles conjugated to high-affine anti-transferrin rece...

Drug Delivery to the Brain

Communications Biology, 2021

Precise methods for quantifying drug accumulation in brain tissue are currently very limited, cha... more Precise methods for quantifying drug accumulation in brain tissue are currently very limited, challenging the development of new therapeutics for brain disorders. Transcardial perfusion is instrumental for removing the intravascular fraction of an injected compound, thereby allowing for ex vivo assessment of extravasation into the brain. However, pathological remodeling of tissue microenvironment can affect the efficiency of transcardial perfusion, which has been largely overlooked. We show that, in contrast to healthy vasculature, transcardial perfusion cannot remove an injected compound from the tumor vasculature to a sufficient extent leading to considerable overestimation of compound extravasation. We demonstrate that 3D deep imaging of optically cleared tumor samples overcomes this limitation. We developed two machine learning-based semi-automated image analysis workflows, which provide detailed quantitative characterization of compound extravasation patterns as well as tumor a...

American Journal of Hematology, 2017

transferrin receptor antibody targeted gold nanoparticles DTU Orbit (12/11/2019) Modulating antib... more transferrin receptor antibody targeted gold nanoparticles DTU Orbit (12/11/2019) Modulating antibody affinity towards the transferrin receptor to increase brain uptake of anti-transferrin receptor antibody targeted gold nanoparticles Drug delivery to the brain is hampered by the presence of the blood-brain barrier (BBB) that under physiological conditions precludes entrance of most substances contained in the systemic circulation. Thus, this barrier must be overcome to deliver medicines into the brain parenchyma. The transferrin receptor is exclusively expressed on capillaries of the brain, which makes it an interesting target for transport of drugs towards the brain. However, the current evidence on the receptor movement in brain capillaries does not suggest transcytosis, and delivering medicines or nanoparticles using antibodies towards this receptor has largely been without success. We investigated the impact of antibody affinity on the transport of gold nanoparticles into the br...

Ultrasound in Medicine & Biology, 2021

The blood-brain barrier (BBB) is a major obstacle to treating several brain disorders. Focused ul... more The blood-brain barrier (BBB) is a major obstacle to treating several brain disorders. Focused ultrasound (FUS) in combination with intravascular microbubbles increases BBB permeability by opening tight junctions, creating endothelial cell openings, improving endocytosis and increasing transcytosis. Here we investigated whether combining FUS and microbubbles with transferrin receptor-targeting liposomes would result in enhanced delivery to the brain of post-natal rats compared with liposomes lacking the BBB-targeting moiety. For all animals, increased BBB permeability was observed after FUS treatment. A 40% increase in accumulation of transferrin receptor-targeting liposomes was observed in the FUS-treated hemisphere, whereas the isotype immunoglobulin G liposomes showed no increased accumulation. Confocal laser scanning microscopy of brain sections revealed that both types of liposomes were mainly observed in endothelial cells in the FUS-treated hemisphere. The results demonstrate that FUS and microbubble treatment combined with BBB-targeting liposomes could be a promising approach to enhance drug delivery to the brain.

The FEBS Journal, 2021

Brain homeostasis depends on the existence of the blood–brain barrier (BBB). Despite decades of r... more Brain homeostasis depends on the existence of the blood–brain barrier (BBB). Despite decades of research, the factors and signalling pathways for modulating and maintaining BBB integrity are not fully elucidated. Here, we characterise the expression and function of the multifunctional receptor, sortilin, in the cells of the BBB, in vivo and in vitro. We show that sortilin acts as an important regulatory protein of the BBB’s tightness. In rats lacking sortilin, the BBB was leaky, which correlated well with relocated distribution of the localisation of zonula occludens‐1, VE‐cadherin and β‐catenin junctional proteins. Furthermore, the absence of sortilin in brain endothelial cells resulted in decreased phosphorylation of Akt signalling protein and increased the level of phospho‐ERK1/2. As a putative result of MAPK/ERK pathway activity, the junctions between the brain endothelial cells were disintegrated and the integrity of the BBB became compromised. The identified barrier differences between wild‐type and Sort1−/− brain endothelial cells can pave the way for a better understanding of sortilin’s role in the healthy and diseased BBB.

Nature Communications, 2021

Effective treatments of neurodegenerative diseases require drugs to be actively transported acros... more Effective treatments of neurodegenerative diseases require drugs to be actively transported across the blood-brain barrier (BBB). However, nanoparticle drug carriers explored for this purpose show negligible brain uptake, and the lack of basic understanding of nanoparticle-BBB interactions underlies many translational failures. Here, using two-photon microscopy in mice, we characterize the receptor-mediated transcytosis of nanoparticles at all steps of delivery to the brain in vivo. We show that transferrin receptor-targeted liposome nanoparticles are sequestered by the endothelium at capillaries and venules, but not at arterioles. The nanoparticles move unobstructed within endothelium, but transcytosis-mediated brain entry occurs mainly at post-capillary venules, and is negligible in capillaries. The vascular location of nanoparticle brain entry corresponds to the presence of perivascular space, which facilitates nanoparticle movement after transcytosis. Thus, post-capillary venule...

Molecular Neurobiology, 2020

Glioblastoma (GBM) is the most frequent and devastating primary tumor of the central nervous syst... more Glioblastoma (GBM) is the most frequent and devastating primary tumor of the central nervous system with a median survival of 12 to 15 months after diagnosis. GBM is highly difficult to treat due to its delicate location, inter-and intra-tumoral heterogeneity, and high plasticity in response to treatment. In this study, we intracranially implanted primary GBM cells into mice which underwent conventional GBM treatments, including irradiation, temozolomide, and a combination. We obtained single cell suspensions through a combination of mechanical and enzymatic dissociation of brain tissue and investigated in detail the changes in GBM cells in response to conventional treatments in vivo using multi-color flow cytometry and cluster analysis. CD44 expression was elevated in all treatment groups, which was confirmed by subsequent immunohistochemistry. High CD44 expression was furthermore shown to correlate with poor prognosis of GBM and low-grade glioma (LGG) patients. Together, these results indicate a key role for CD44 in glioma pathogenesis.

SUMMARYTreatments of neurodegenerative diseases require biologic drugs to be actively transported... more SUMMARYTreatments of neurodegenerative diseases require biologic drugs to be actively transported across the blood-brain barrier (BBB). To answer outstanding questions regarding transport mechanisms, we determined how and where transcytosis occurs at the BBB. Using two-photon microscopy, we characterized the transport of therapeutic nanoparticles at all steps of delivery to the brain and at the nanoscale resolution in vivo. Transferrin receptor-targeted nanoparticles were taken up by endothelium at capillaries and venules, but not at arterioles. The nanoparticles moved unobstructed within endothelial cells, but transcytosis across the BBB occurred only at post-capillary venules, where endothelial and glial basement membranes form a perivascular space that can accommodate biologics. In comparison, transcytosis was absent in capillaries with closely apposed basement membranes. Thus, post-capillary venules, not capillaries, provide an entry point for transport of large molecules across...

Cancer and Metastasis Reviews, 2020

Cancer treatment remains a challenge due to a high level of intra- and intertumoral heterogeneity... more Cancer treatment remains a challenge due to a high level of intra- and intertumoral heterogeneity and the rapid development of chemoresistance. In the brain, this is further hampered by the blood-brain barrier that reduces passive diffusion of drugs to a minimum. Tumors grow invasively and form new blood vessels, also in brain tissue where remodeling of pre-existing vasculature is substantial. The cancer-associated vessels in the brain are considered leaky and thus could facilitate the transport of chemotherapeutic agents. Yet, brain tumors are extremely difficult to treat, and, in this review, we will address how different aspects of the vasculature in brain tumors contribute to this.

Progress in Neurobiology, 2019

Targeting of the transferrin receptor is one of the most efficient ways of obtaining brain drug d... more Targeting of the transferrin receptor is one of the most efficient ways of obtaining brain drug delivery.  Despite decades of development, very little clinical progression has been made, although few clinical trials are beginning to emerge.  New innovations in terms of modifications of the antibody binding mode has yielded impressive leaps forward in our understanding of the transport process.  Many parameters in the transferrin receptor-mediated transport across the blood-brain barrier are still poorly understood.  Increased understanding of factors like intracellular sorting and subsequent long-term brain retention of TfR-targeted drug delivery systems is needed to fulfill the ambition of clinical use.

Journal of Cell Communication and Signaling, 2019

Exosomes were first described as waste carriers implicated in reticulocyte maturation but has dur... more Exosomes were first described as waste carriers implicated in reticulocyte maturation but has during the past decade been associated with many other cellular functions. The biogenesis of exosomes has been extensively studied and several protein machineries have been identified to dictate their production and release. The newly discovered branches of the autophagy system implicate secretion of waste in endosomal-derived vesicles as is thought for exosome release. Many of the proteins that have been identified as responsible for the formation and release of these vesicles are the same as those identified in exosome biogenesis. In this Perspective, we discuss the possibility of exosomes being a part of the autophagy machinery and the consequences this could have on interpretation of exosome functions.

Journal of Controlled Release, 2019

Systematic review of targeted extracellular vesicles for drug delivery Considerations on methodol... more Systematic review of targeted extracellular vesicles for drug delivery Considerations on methodological and biological heterogeneity

Experimental Cell Research, 2019

A tumorsphere model of glioblastoma multiforme with intratumoral heterogeneity for quantitative a... more A tumorsphere model of glioblastoma multiforme with intratumoral heterogeneity for quantitative analysis of cellular migration and drug response

Journal of Controlled Release, 2019

Transport of the majority of therapeutic molecules to the brain is precluded by the presence of t... more Transport of the majority of therapeutic molecules to the brain is precluded by the presence of the blood-brain barrier (BBB) rendering efficient treatment of many neurological disorders impossible. This BBB, nonetheless, may be circumvented by targeting receptors and transport proteins expressed on the luminal surface of the brain capillary endothelial cells (BCECs). The transferrin receptor (TfR) has remained a popular target since its original description for this purpose, although clinical progression of TfR-targeted drug constructs or nanomedicines remains unsuccessful. One proposed issue pertaining to the use of TfR-targeting in nanomedicines is the efficient tuning of the ligand density on the nanoparticle surface. We studied the impact of TfR antibody density on the uptake and transport of nanoparticles into the brain, taking a parallel approach to investigate the impact on both antibody-functionalized gold nanoparticles (AuNPs) and cargo-loaded liposomes. We report that among three different low-range mean ligand densities (0.15, 0.3, and 0.6 * 10 3 antibodies/μm 2), the highest density yielded the highest ability towards both targeting of the BCECs and subsequent transport across the BBB in vivo, and in vitro using primary cultures of the murine BBB. We also find that TfR-targeting on liposomes in the mouse may induce severe adverse effects after intravenous administration.

Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2018

Circulating biomarkers have a great potential in diagnosing cancer diseases at early stages, wher... more Circulating biomarkers have a great potential in diagnosing cancer diseases at early stages, where curative treatment is a realistic possibility. In the recent years, using extracellular vesicles (EVs) derived from blood as biomarkers has gained widespread popularity, mainly because they are thought to be easy to isolate and carry a vast variety of biological cargos that can be analyzed for biomarker purposes. However, our current knowledge on the plasma EV concentration in normophysiological states is sparse. Here, we provide the very first mean estimate of the plasma EV concentration based on values obtained from a thorough literature review. The different estimates obtained from the literature are correlated to the isolation techniques used to obtain them, illustrating how some methodologies may over-or underestimate the plasma EV concentration. We also show that the estimated plasma EV concentration (approximately 10 10 EVs per mL) defines EVs as a minority population compared to other colloidal particles of the systemic circulation, namely the lipoproteins, which are known contaminants in EV isolates and carry biomarker molecules themselves. Lastly, we introduce the possibility of regarding EVs and lipoproteins as a continuum of lipid-containing particles to which biomarker molecules can be associated. Using such a holistic approach, increased strength of plasma-derived cancer biomarkers may soon be revealed.