Extracellular Vesicle-DNA: The Next Liquid Biopsy Biomarker for Early Cancer Diagnosis?

After a short introduction about the history of liquid biopsy, aimed to noninvasively replace the common tissue biopsy as a help for cancer diagnosis, this review is focused on extracellular vesicles (EVs), as the main third component, which is now coming into the light of liquid biopsy. Cell-derived EV release is a recently discovered general cellular property, and EVs harbor many cellular components reflecting their cell of origin. This is also the case for tumoral cells, and their cargoes might therefore be a "treasure chest" for cancer biomarkers. This has been extensively explored for a decade, but the EV-DNA content escaped this worldwide query until recently. The aim of this review is to gather the pilot studies focused on the DNA content of circulating cell-derived EVs, and the following five years of studies about the circulating tumor EV-DNA. The recent preclinical studies about the circulating tEV-derived gDNA as a potential cancer biomarker developed into a puzzling controversy about the presence of DNA into exosomes, coupled with an increased unexpected non vesicular complexity of the extracellular environment. This is discussed in the present review, together with the challenges that need to be solved before any efficient clinical transfer of EV-DNA as a quite promising cancer diagnosis biomarker.

Current Search through Liquid Biopsy of Effective Biomarkers for Early Cancer Diagnosis into the Rich Cargoes of Extracellular Vesicles.

There exist many different human cancers, but regardless of the cancer type, an early diagnosis is a necessary condition for further optimal outcomes from the disease. Therefore, efficient specific and sensitive cancer biomarkers are urgently needed. This is especially true for the cancers depicting a silent progression, and those only diagnosed in an already metastatic state with a poor survival prognostic. After a rapid overview of the previous methods for cancer diagnosis, the outstanding characteristics of extracellular vesicles (EVs) will be presented, as new interesting candidates for early cancer diagnosis in human biofluid non-invasive liquid biopsy. The present review aims to give the state-of-the-art of the numerous searches of efficient EV-mediated cancer diagnosis. The corresponding literature quest was performed by means of an original approach, using a powerful Expernova Questel big data platform, which was specifically adapted for a literature search on EVs. The chosen collected scientific papers are presented in two parts, the first one drawing up a picture of the current general status of EV-mediated cancer diagnosis and the second one showing recent applications of such EV-mediated diagnosis for six important human-specific cancers, i.e., lung, breast, prostate, colorectal, ovary and pancreatic cancers. However, the promising perspective of finally succeeding in the worldwide quest for the much-needed early cancer diagnosis has to be moderated by the many remaining challenges left to solve before achieving the efficient clinical translation of the constantly increasing scientific knowledge.

Dictyostelium: A Model for Studying the Extracellular Vesicle Messengers Involved in Human Health and Disease.

Cell-derived extracellular vesicles (EVs) are newly uncovered messengers for intercellular communication. They are released by almost all cell types in the three kingdoms, Archeabacteria, Bacteria and Eukaryotes. They are known to mediate important biological functions and to be increasingly involved in cell physiology and in many human diseases, especially in oncology. The aim of this review is to recapitulate the current knowledge about EVs and to summarize our pioneering work about Dictyostelium discoideum EVs. However, many challenges remain unsolved in the EV research field, before any EV application for theranostics (diagnosis, prognosis, and therapy) of human cancers, can be efficiently implemented in the clinics. Dictyostelium might be an outstanding eukaryotic cell model for deciphering the utmost challenging problem of EV heterogeneity, and for unraveling the still mostly unknown mechanisms of their specific functions as mediators of intercellular communication.

Raman tweezers microspectroscopy of circa 100 nm extracellular vesicles.

The technique of Raman tweezers microspectroscopy (RTM) for the global biomolecular content characterization of a single extracellular vesicle (EV) or a small number of EVs or other nanoscale bioparticles in an aqueous dispersion in the difficult-to-access size range of near 100 nm is described in detail. The particularities and potential of RTM are demonstrated using the examples of DOPC liposomes, exosomes from human urine and rat hepatocytes, and a mixed sample of the transfection reagent FuGENE in diluted DNA solution. The approach of biomolecular component analysis for the estimation of the main biomolecular contributions (proteins, lipids, nucleic acids, carotenoids, etc.) is proposed and discussed. Direct Raman evidence for strong intra-sample biomolecular heterogeneity of individual optically trapped EVs, due to variable contributions from nucleic acids and carotenoids in some preparations, is reported. On the basis of the results obtained, we are making an attempt to convince the scientific community that RTM is a promising method of single-EV research; to our knowledge, it is the only technique available at the moment that provides unique information about the global biomolecular composition of a single vesicle or a small number of vesicles, thus being capable of unravelling the high diversity of EV subpopulations, which is one of the most significant urgent challenges to overcome. Possible RTM applications include, among others, searching for DNA biomarkers, cancer diagnosis, and discrimination between different subpopulations of EVs, lipid bodies, protein aggregates and viruses.

Assets of the non-pathogenic microorganism Dictyostelium discoideum as a model for the study of eukaryotic extracellular vesicles.

Dictyostelium discoideum microvesicles have recently been presented as a valuable model for eukaryotic extracellular vesicles. Here, the advantages of D. discoideum for unraveling important biological functions of extracellular vesicles in general are detailed. D. discoideum, a non-pathogenic eukaryotic microorganism, belongs to a billion-year-old Amoeboza lineage, which diverged from the animal-fungal lineage after the plant animal-split. During growth and early starvation-induced development, it presents analogies with lymphocytes and macrophages with regard to motility and phagocytosis capability, respectively. Its 6-chromosome genome codes for about 12,500 genes, some showing analogies with human genes. The presence of extracellular vesicles during cell growth has been evidenced as a detoxification mechanism of various structurally unrelated drugs. Controls led to the discovery of constitutive extracellular vesicle secretion in this microorganism, which was an important point. It means that the secretion of extracellular vesicles occurs, in the absence of any drug, during both cell growth and early development. This constitutive secretion of D. discoideum cells is very likely to play a role in intercellular communication. The detoxifying secreted vesicles, which can transport drugs outside the cells, can also act as "Trojan horses", capable of transferring these drugs not only into naïve D. discoideum cells, but into human cells as well. Therefore, these extracellular vesicles were proposed as a new biological drug delivery tool. Moreover, Dictyostelium, chosen by the NIH (USA) as a new model organism for biomedical research, has already been used for studying some human diseases. These cells, which are much easier to manipulate than human cells, can be easily designed in simple conditioned medium experiments. Owing to the increasing consensus that extracellular vesicles are probably important mediators of intercellular communication, D. discoideum is here suggested to constitute a convenient model for tracking as yet unknown biological functions of eukaryotic extracellular vesicles.

Assets of the non-pathogenic microorganism Dictyostelium discoideum as a model for the study of eukaryotic extracellular vesicles.

Dictyostelium discoideum microvesicles have recently been presented as a valuable model for eukaryotic extracellular vesicles. Here, the advantages of D. discoideum for unraveling important biological functions of extracellular vesicles in general are detailed. D. discoideum, a non-pathogenic eukaryotic microorganism, belongs to a billion-year-old Amoeboza lineage, which diverged from the animal-fungal lineage after the plant animal-split. During growth and early starvation-induced development, it presents analogies with lymphocytes and macrophages with regard to motility and phagocytosis capability, respectively. Its 6-chromosome genome codes for about 12,500 genes, some showing analogies with human genes. The presence of extracellular vesicles during cell growth has been evidenced as a detoxification mechanism of various structurally unrelated drugs. Controls led to the discovery of constitutive extracellular vesicle secretion in this microorganism, which was an important point. It means that the secretion of extracellular vesicles occurs, in the absence of any drug, during both cell growth and early development. This constitutive secretion of D. discoideum cells is very likely to play a role in intercellular communication. The detoxifying secreted vesicles, which can transport drugs outside the cells, can also act as "Trojan horses", capable of transferring these drugs not only into naïve D. discoideum cells, but into human cells as well. Therefore, these extracellular vesicles were proposed as a new biological drug delivery tool. Moreover, Dictyostelium, chosen by the NIH (USA) as a new model organism for biomedical research, has already been used for studying some human diseases. These cells, which are much easier to manipulate than human cells, can be easily designed in simple conditioned medium experiments. Owing to the increasing consensus that extracellular vesicles are probably important mediators of intercellular communication, D. discoideum is here suggested to constitute a convenient model for tracking as yet unknown biological functions of eukaryotic extracellular vesicles.

Cell-derived microvesicles and antitumoral multidrug resistance.

Antitumoral chemotherapeutic treatments are often impaired by innate or acquired multidrug resistance (MDR). After four decades of MDR research, having underlined its complexity, new knowledge about the mechanisms of tumor resistance to antineoplastic drugs is a prerequisite for improving chemotherapy. Following our observations with a non-pathogenic eukaryotic microorganism, Dictyostelium discoideum, I suggest that MDR in tumor cells might be the consequence of a detoxification mechanism, mediated by cell-derived microvesicles. Recently published observations with tumoral human cells support this hypothesis. First, these cell-derived vesicles might impair chemotherapeutic efficiency of many structurally-different antineoplastic agents by preventing them to reach their intracellular target, followed by their expulsion outside the tumor cells, as observed for Dictyostelium cells. Secondly, besides their newly recognized function of intercellular communication, the cell-derived vesicles might also act as intercellular transporters of multidrug resistance proteins. Experiments are suggested for checking the hypothesis of cell-derived vesicles mediating multidrug resistance.

Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy.

The joint use of 3 complementary techniques, namely, nanoparticle tracking analysis (NTA), cryo-electron microscopy (Cryo-EM) and Raman tweezers microspectroscopy (RTM), is proposed for a rapid characterisation of extracellular vesicles (EVs) of various origins. NTA is valuable for studying the size distribution and concentration, Cryo-EM is outstanding for the morphological characterisation, including observation of vesicle heterogeneity, while RTM provides the global chemical composition without using any exogenous label. The capabilities of this approach are evaluated on the example of cell-derived vesicles of Dictyostelium discoideum, a convenient general model for eukaryotic EVs. At least 2 separate species differing in chemical composition (relative amounts of DNA, lipids and proteins, presence of carotenoids) were found for each of the 2 physiological states of this non-pathogenic microorganism, that is, cell growth and starvation-induced aggregation. These findings demonstrate the specific potency of RTM. In addition, the first Raman spectra of human urinary exosomes are reported, presumably constituting the primary step towards Raman characterisation of EVs for the purpose of human diseases diagnoses.

Nanovesicles released by Dictyostelium cells: a potential carrier for drug delivery.

Nanovesicles released by Dictyostelium discoideum cells grown in the presence of the DNA-specific dye Hoechst 33342 have been previously shown to mediate the transfer of the dye into the nuclei of Hoechst-resistant cells. The present investigation extends this work by conducting experiments in the presence of hypericin, a fluorescent therapeutic photosensitizer assayed for antitumoral photodynamic therapy. Nanovesicles released by Dictyostelium cells exhibit an averaged diameter between 50 and 150 nm, as measured by transmission cryoelectron microscopy. A proteomic analysis reveals a predominance of actin and actin-related proteins. The detection of a lysosomal membrane protein (LIMP II) indicates that these vesicles are likely generated in the late endosomal compartment. The use of the hypericin-containing nanovesicles as nanodevices for in vitro drug delivery was investigated by fluorescence microscopy. The observed signal was almost exclusively located in the perinuclear area of two human cell lines, skin fibroblasts (HS68) and cervix carcinoma (HeLa) cells. Studies by confocal microscopy with specific markers of cell organelles, provided evidence that hypericin was accumulated in the Golgi apparatus. All these data shed a new light on in vitro drug delivery by using cell-released vesicles as carriers.

Dictyostelium extracellular vesicles containing hoechst 33342 transfer the dye into the nuclei of living cells: a fluorescence study.

Cells of the eukaryotic unicellular microorganism Dictyostelium discoideum are constitutively resistant to vital staining of their nuclei by the DNA-specific dye Hoechst 33342. By studying the mechanisms of this resistance, we evidenced that these cells expel vesicles containing the dye for detoxification (Tatischeff et al., Cell Mol Life Sci, 54: 476-87, 1998). The question to be addressed in the present work is the potential use of these extracellular vesicles as a biological drug delivery tool, using Hoechst 33342 as a model of a DNA-targeting drug. After cell growth with or without the dye, vesicles were prepared from the cell-free growth medium by differential centrifugation, giving rise to two types of vesicles. Negative staining electron microscopy showed their large heterogeneity in size. Using fluorescence techniques, data were obtained on the dye loading and its environment inside the vesicles. By UV video-microscopy, it was demonstrated that the dye-containing vesicles were able to deliver it into the nuclei of naive Dictyostelium cells, thus overcoming their constitutive resistance to the free dye. A vesicle-mediated dye-transfer into the nuclei of living human leukaemia multidrug resistant K562r cells was also observed.