Glycan Structures in Osteosarcoma as Targets for Lectin-Based Chimeric Antigen Receptor Immunotherapy.

Osteosarcoma is a type of bone cancer that primarily affects children and young adults. The overall 5-year survival rate for localized osteosarcoma is 70-75%, but it is only 20-30% for patients with relapsed or metastatic tumors. To investigate potential glycan-targeting structures for immunotherapy, we stained primary osteosarcomas with recombinant C-type lectin CD301 (MGL, CLEC10A) and observed moderate to strong staining on 26% of the tumors. NK92 cells expressing a CD301-CAR recognized and eliminated osteosarcoma cells in vitro. Cytotoxic activity assays correlated with degranulation and cytokine release assays. Combination with an inhibitory antibody against the immune checkpoint TIGIT (T-cell immunoreceptor with lg and ITIM domains) showed promising additional effects. Overall, this study showed, for the first time, the expression of CD301 ligands in osteosarcoma tissue and demonstrated their use as potential target structures for lectin-based immunotherapy.

Multimodal cartography of human lymphopoiesis reveals B and T/NK/ILC lineages are subjected to differential regulation.

The developmental cartography of human lymphopoiesis remains incompletely understood. Here, we establish a multimodal map demonstrating that lymphoid specification follows independent direct or stepwise hierarchic routes converging toward the emergence of newly characterized CD117lo multi-lymphoid progenitors (MLPs) that undergo a proliferation arrest before entering the CD127- (NK/ILC/T) or CD127+ (B) lymphoid pathways. While the differentiation of CD127- early lymphoid progenitors is mainly driven by Flt3 signaling, emergence of their CD127+ counterparts is regulated cell-intrinsically and depends exclusively on the divisional history of their upstream precursors, including hematopoietic stem cells. Further, transcriptional mapping of differentiation trajectories reveals that whereas myeloid granulomonocytic lineages follow continuous differentiation pathways, lymphoid trajectories are intrinsically discontinuous and characterized by sequential waves of cell proliferation allowing pre-commitment amplification of lymphoid progenitor pools. Besides identifying new lymphoid specification pathways and regulatory checkpoints, our results demonstrate that NK/ILC/T and B lineages are under fundamentally distinct modes of regulation. (149 words).

Glyco-binding domain chimeric antigen receptors as a new option for cancer immunotherapy.

In the last decade, treatment using Chimeric Antigen Receptor (CAR) are largely studied and demonstrate the potential of immunotherapeutic strategies, as seen mainly for blood related cancers. Still, efficient CAR-T cell approaches especially for the treatment of solid tumors are needed. Tn- and Sialyl-Tn antigens are tumor associated carbohydrate antigens correlating with poor prognosis and tumor metastasis on a variety of tumor entities. These glycans can be recognized by CD301 (CLEC10A, MGL), which is a surface receptor found primarily on immune cells. In the present study, we hypothesized, that it is possible to use newly generated CD301-bearing CARs, enabling cytotoxic effector cells to recognize and eliminate breast cancer cells. Thus, we genetically modified human NK92 cells with different chimeric receptors based on the carbohydrate recognition domain (CRD) of human CD301. We assessed their cytotoxic activity in vitro demonstrating the specific recognition of CD301 ligand positive cell lines. These results were confirmed by degranulation assays and in cytokine release assays. Overall, this study demonstrates CD301-CARs represent a cost-effective and fast alternative to conventional scFv CARs for cancer immunotherapy.

MAP kinase activating death domain deficiency is a novel cause of impaired lymphocyte cytotoxicity.

Most hereditary forms of hemophagocytic lymphohistiocytosis (HLH) are caused by defects of cytotoxicity, including the vesicle trafficking disorder Griscelli syndrome type 2 (GS2, RAB27A deficiency). Deficiency of the mitogen-activated protein kinase activating death domain protein (MADD) results in a protean syndrome with neurological and endocrinological involvement. MADD acts as a guanine nucleotide exchange factor for small guanosine triphosphatases, including RAB27A. A homozygous splice site mutation in MADD was identified in a female infant with syndromic features, secretory diarrhea, and features of HLH. Aberrant splicing caused by this mutation leads to an in-frame deletion of 30 base pairs and favors other aberrant variants. Patient natural killer (NK) cells and cytotoxic T cells showed a severe degranulation defect leading to absent perforin-mediated cytotoxicity. Platelets displayed defective adenosine triphosphate secretion, similar to that in GS2. To prove causality, we introduced a CRISPR/Cas9-based MADD knockout in the NK cell line NK-92mi. MADD-deficient NK-92mi cells showed a degranulation defect and impaired cytotoxicity similar to that of the patient. The defect of cytotoxicity was confirmed in another patient with MADD deficiency. In conclusion, RAB27A-interacting MADD is involved in vesicle release by cytotoxic cells and platelets. MADD deficiency causes a degranulation defect and represents a novel disease predisposing to an HLH phenotype.

Reduced adhesion of aged intestinal stem cells contributes to an accelerated clonal drift.

Upon aging, the function of the intestinal epithelium declines with a concomitant increase in aging-related diseases. ISCs play an important role in this process. It is known that ISC clonal dynamics follow a neutral drift model. However, it is not clear whether the drift model is still valid in aged ISCs. Tracking of clonal dynamics by clonal tracing revealed that aged crypts drift into monoclonality substantially faster than young ones. However, ISC tracing experiments, in vivo and ex vivo, implied a similar clonal expansion ability of both young and aged ISCs. Single-cell RNA sequencing for 1,920 high Lgr5 ISCs from young and aged mice revealed increased heterogeneity among subgroups of aged ISCs. Genes associated with cell adhesion were down-regulated in aged ISCs. ISCs of aged mice indeed show weaker adhesion to the matrix. Simulations applying a single cell-based model of the small intestinal crypt demonstrated an accelerated clonal drift at reduced adhesion strength, implying a central role for reduced adhesion for affecting clonal dynamics upon aging.

The Reconstitution Dynamics of Cultivated Hematopoietic Stem Cells and Progenitors Is Independent of Age.

Hematopoietic stem cell transplantation (HSCT) represents the only curative treatment option for numerous hematologic malignancies. While the influence of donor age and the composition of the graft have already been examined in clinical and preclinical studies, little information is available on the extent to which different hematological subpopulations contribute to the dynamics of the reconstitution process and on whether and how these contributions are altered with age. In a murine model of HSCT, we therefore simultaneously tracked different cultivated and transduced hematopoietic stem and progenitor cell (HSPC) populations using a multicolor-coded barcode system (BC32). We studied a series of age-matched and age-mismatched transplantations and compared the influence of age on the reconstitution dynamics. We show that reconstitution from these cultured and assembled grafts was substantially driven by hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) independent of age. The reconstitution patterns were polyclonal and stable in all age groups independently of the variability between individual animals, with higher output rates from MPPs than from HSCs. Our experiments suggest that the dynamics of reconstitution and the contribution of cultured and individually transduced HSPC subpopulations are largely independent of age. Our findings support ongoing efforts to expand the application of HSCT in older individuals as a promising strategy to combat hematological diseases, including gene therapy applications.

genBaRcode: a comprehensive R-package for genetic barcode analysis.

MOTIVATION: Genetic barcodes have been established as an efficient method to trace clonal progeny of uniquely labeled cells by introducing artificial genetic sequences into the corresponding genomes. The assessment of those sequences relies on next generation sequencing and the subsequent analysis aiming to identify sequences of interest and correctly quantifying their abundance. RESULTS: We developed the genBaRcode package as a toolbox combining the flexibility of digesting next generation sequencing reads with or without a sophisticated barcode structure, with a variety of error-correction approaches and the availability of several types of visualization routines. Furthermore, a graphical user interface was incorporated to allow also less experienced R users package-based analyses. Finally, the provided tool is intended to bridge the gap between generating and analyzing barcode data and thereby supporting the establishment of standardized and reproducible analysis strategies. AVAILABILITY AND IMPLEMENTATION: The genBaRcode package is available at CRAN (https://cran.r-project.org/package=genBaRcode).

The Actin Binding Protein Plastin-3 Is Involved in the Pathogenesis of Acute Myeloid Leukemia.

Leukemia-initiating cells reside within the bone marrow in specialized niches where they undergo complex interactions with their surrounding stromal cells. We have identified the actin-binding protein Plastin-3 (PLS3) as potential player within the leukemic bone marrow niche and investigated its functional role in acute myeloid leukemia. High expression of PLS3 was associated with a poor overall and event-free survival for AML patients. These findings were supported by functional in vitro and in vivo experiments. AML cells with a PLS3 knockdown showed significantly reduced colony numbers in vitro while the PLS3 overexpression variants resulted in significantly enhanced colony numbers compared to their respective controls. Furthermore, the survival of NSG mice transplanted with the PLS3 knockdown cells showed a significantly prolonged survival in comparison to mice transplanted with the control AML cells. Further studies should focus on the underlying leukemia-promoting mechanisms and investigate PLS3 as therapeutic target.

Engrafted parenchymal brain macrophages differ from microglia in transcriptome, chromatin landscape and response to challenge.

Microglia are yolk sac-derived macrophages residing in the parenchyma of brain and spinal cord, where they interact with neurons and other glial. After different conditioning paradigms and bone marrow (BM) or hematopoietic stem cell (HSC) transplantation, graft-derived cells seed the brain and persistently contribute to the parenchymal brain macrophage compartment. Here we establish that graft-derived macrophages acquire, over time, microglia characteristics, including ramified morphology, longevity, radio-resistance and clonal expansion. However, even after prolonged CNS residence, transcriptomes and chromatin accessibility landscapes of engrafted, BM-derived macrophages remain distinct from yolk sac-derived host microglia. Furthermore, engrafted BM-derived cells display discrete responses to peripheral endotoxin challenge, as compared to host microglia. In human HSC transplant recipients, engrafted cells also remain distinct from host microglia, extending our finding to clinical settings. Collectively, our data emphasize the molecular and functional heterogeneity of parenchymal brain macrophages and highlight potential clinical implications for HSC gene therapies aimed to ameliorate lysosomal storage disorders, microgliopathies or general monogenic immuno-deficiencies.

A track of the clones: new developments in cellular barcoding.

International experts from multiple disciplines gathered at Homerton College in Cambridge, UK from September 12-14, 2018 to consider recent advances and emerging opportunities in the clonal tracking of hematopoiesis in one of a series of StemCellMathLab workshops. The group included 35 participants with experience in the fields of theoretical and experimental aspects of clonal tracking, and ranged from doctoral students to senior professors. Data from a variety of model systems and from clinical gene therapy trials were discussed, along with strategies for data analysis and sharing and challenges arising due to underlying assumptions in data interpretation and communication. Recognizing the power of this technology underpinned a group consensus of a need for improved mechanisms for sharing data and analytical protocols to maintain reproducibility and rigor in its application to complex tissues.

Cellular Barcoding Identifies Clonal Substitution as a Hallmark of Local Recurrence in a Surgical Model of Head and Neck Squamous Cell Carcinoma.

Local recurrence after surgery for head and neck squamous cell carcinoma (HNSCC) remains a common event associated with a dismal prognosis. Improving this outcome requires a better understanding of cancer cell populations that expand from postsurgical minimal residual disease (MRD). Therefore, we assessed clonal dynamics in a surgical model of barcoded HNSCC growing in the submental region of immunodeficient mice. Clonal substitution and massive reduction of clonal heterogeneity emerged as hallmarks of local recurrence, as the clones dominating in less heterogeneous recurrences were scarce in their matched primary tumors. These lineages were selected by their ability to persist after surgery and competitively expand from MRD. Clones enriched in recurrences exhibited both private and shared genetic features and likely originated from ancestors shared with clones dominating in primary tumors. They demonstrated high invasiveness and epithelial-to-mesenchymal transition, eventually providing an attractive target for obtaining better local control for these tumors.

Lysosomal Proteome and Secretome Analysis Identifies Missorted Enzymes and Their Nondegraded Substrates in Mucolipidosis III Mouse Cells.

Targeting of soluble lysosomal enzymes requires mannose 6-phosphate (M6P) signals whose formation is initiated by the hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2ß2γ2). Upon proteolytic cleavage by site-1 protease, the α/ß-subunit precursor is catalytically activated but the functions of γ-subunits (Gnptg) in M6P modification of lysosomal enzymes are unknown. To investigate this, we analyzed the Gnptg expression in mouse tissues, primary cultured cells, and in Gnptg reporter mice in vivo, and found high amounts in the brain, eye, kidney, femur, vertebra and fibroblasts. Consecutively we performed comprehensive quantitative lysosomal proteome and M6P secretome analysis in fibroblasts of wild-type and Gnptgko mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/ß-precursor was not affected by Gnptg deficiency, the GlcNAc-1-phosphotransferase activity was significantly reduced. We purified lysosomes and identified 29 soluble lysosomal proteins by SILAC-based mass spectrometry exhibiting differential abundance in Gnptgko fibroblasts which was confirmed by Western blotting and enzymatic activity analysis for selected proteins. A subset of these lysosomal enzymes show also reduced M6P modifications, fail to reach lysosomes and are secreted, among them α-l-fucosidase and arylsulfatase B. Low levels of these enzymes correlate with the accumulation of non-degraded fucose-containing glycostructures and sulfated glycosaminoglycans in Gnptgko lysosomes. Incubation of Gnptgko fibroblasts with arylsulfatase B partially rescued glycosaminoglycan storage. Combinatorial treatments with other here identified missorted enzymes of this degradation pathway might further correct glycosaminoglycan accumulation and will provide a useful basis to reveal mechanisms of selective, Gnptg-dependent formation of M6P residues on lysosomal proteins.

High Bone Turnover in Mice Carrying a Pathogenic Notch2 Mutation Causing Hajdu-Cheney Syndrome.

Hajdu-Cheney syndrome (HCS) is a rare autosomal-dominant disorder primarily characterized by acro-osteolysis and early-onset osteoporosis. Genetically, HCS is caused by nonsense or deletion mutations within exon 34 of the NOTCH2 gene, resulting in premature translational termination and production of C-terminally truncated NOTCH2 proteins that are predicted to activate NOTCH2-dependent signaling. To understand the role of Notch2 in bone remodeling, we developed a mouse model of HCS by introducing a pathogenic mutation (6272delT) into the murine Notch2 gene. By µCT and undecalcified histology, we observed generalized osteopenia in two independent mouse lines derived by injection of different targeted embryonic stem (ES) cell clones, yet acro-osteolysis did not occur until the age of 52 weeks. Cellular and dynamic histomorphometry revealed a high bone turnover situation in Notch2+/HCS mice, since osteoblast and osteoclast indices were significantly increased compared with wild-type littermates. Whereas ex vivo cultures failed to uncover cell-autonomous gain-of-functions within the osteoclast or osteoblast lineage, an unbiased RNA sequencing approach identified Tnfsf11 and Il6 as Notch-signaling target genes in bone marrow cells cultured under osteogenic conditions. Because we further observed that the high-turnover pathology of Notch2+/HCS mice was fully normalized by alendronate treatment, our results demonstrate that mutational activation of Notch2 does not directly control osteoblast activity but favors a pro-osteoclastic gene expression pattern, which in turn triggers high bone turnover. © 2017 American Society for Bone and Mineral Research.

Enzymatic characterization of novel arylsulfatase A variants using human arylsulfatase A-deficient immortalized mesenchymal stromal cells.

Metachromatic leukodystrophy (MLD) is an autosomal-recessive lysosomal storage disease caused by mutations in the ARSA gene leading to arylsulfatase A (ARSA) deficiency and causing sulfatide accumulation. Main symptoms of the disease are progressive demyelination, neurological dysfunction, and reduced life expectancy. To date, more than 200 different ARSA variants have been reported in MLD patients. Here, we report the biochemical characterization of seven novel pathogenic variants (c.98T > C, c.195delC, c.229G > C, c.545C > G, c.674A > G, c.852T > A, and c.1274A > G), which were found when sequencing a cohort of 31 German MLD families. For that purpose, the ARSA cDNAs carrying the respective mutations inserted by site-directed mutagenesis were cloned into a MigR1 (MSCV, IRES, GFP, retrovirus-1) vector. The constructs were overexpressed using retroviral gene transfer in immortalized, human multipotent mesenchymal stromal cells prepared from a patient deficient in ARSA activity (late infantile MLD). In this novel ARSA-/- cell system, the seven ARSA mutants showed ARSA activity of less than 10% when compared with wild type, which is evidence for the pathogenicity of all seven variants. In conclusion, the system of ARSA-/- -immortalized MSC turned out to be a helpful novel tool for the biochemical characterization of ARSA variants.

Genetic Barcodes Facilitate Competitive Clonal Analyses In Vivo.

Monitoring the fate of individual cell clones is an important task to better understand normal tissue regeneration, for example after hematopoietic stem cell (HSC) transplantation, but also cancerogenesis. Based on their integration into the host cell's genome, retroviral vectors are commonly used to stably mark target cells and their progeny. The development of genetic barcoding techniques has opened new possibilities to determine clonal composition and dynamics in great detail. A modular genetic barcode was recently introduced consisting of 32 variable positions (BC32) with a customized backbone, and its advantages were demonstrated with regard to barcode calling and quantification. The study presented applied the BC32 system in a complex in vivo situation, namely to analyze clonal reconstitution dynamics for HSC grafts consisting of up to three cell populations with distinguishable barcodes using different alpha- and lentiviral vectors. In a competitive transplantation setup, it was possible to follow the differently marked cell populations within individual animals. This enabled the clonal contribution of the different BC32 constructs during reconstitution and long-term hematopoiesis in the peripheral blood and the spatial distribution in bone marrow and spleen to be identified. Thus, it was demonstrated that the system allows the output of individually marked cells to be tracked in vivo and their influence on clonal dynamics to be analyzed. Successful application of the BC32 system in a complex, competitive in vivo situation provided proof-of-principle that its high complexity and the large Hamming distance between individual barcodes, combined with the easy customization, facilitate efficient and precise quantification, even without prior knowledge of individual barcode sequences. Importantly, simultaneous high-sensitivity analyses of different cell populations in single animals may significantly reduce numbers of animals required to investigate specific scientific questions in accordance with RRR principles. It is concluded that this BC32 system will be excellently suited for various research applications in regenerative medicine and cancer biology.

Clonal competition in BcrAbl-driven leukemia: how transplantations can accelerate clonal conversion.

BACKGROUND: Clonal competition in cancer describes the process in which the progeny of a cell clone supersedes or succumbs to other competing clones due to differences in their functional characteristics, mostly based on subsequently acquired mutations. Even though the patterns of those mutations are well explored in many tumors, the dynamical process of clonal selection is underexposed. METHODS: We studied the dynamics of clonal competition in a BcrAbl-induced leukemia using a γ-retroviral vector library encoding the oncogene in conjunction with genetic barcodes. To this end, we studied the growth dynamics of transduced cells on the clonal level both in vitro and in vivo in transplanted mice. RESULTS: While we detected moderate changes in clonal abundancies in vitro, we observed monoclonal leukemias in 6/30 mice after transplantation, which intriguingly were caused by only two different BcrAbl clones. To analyze the success of these clones, we applied a mathematical model of hematopoietic tissue maintenance, which indicated that a differential engraftment capacity of these two dominant clones provides a possible explanation of our observations. These findings were further supported by additional transplantation experiments and increased BcrAbl transcript levels in both clones. CONCLUSION: Our findings show that clonal competition is not an absolute process based on mutations, but highly dependent on selection mechanisms in a given environmental context.

Limitations and challenges of genetic barcode quantification.

Genetic barcodes are increasingly used to track individual cells and to quantitatively assess their clonal contributions over time. Although barcode quantification relies entirely on counting sequencing reads, detailed studies about the method's accuracy are still limited. We report on a systematic investigation of the relation between barcode abundance and resulting read counts after amplification and sequencing using cell-mixtures that contain barcodes with known frequencies ("miniBulks"). We evaluated the influence of protocol modifications to identify potential sources of error and elucidate possible limitations of the quantification approach. Based on these findings we designed an advanced barcode construct (BC32) to improved barcode calling and quantification, and to ensure a sensitive detection of even highly diluted barcodes. Our results emphasize the importance of using curated barcode libraries to obtain interpretable quantitative data and underline the need for rigorous analyses of any utilized barcode library in terms of reliability and reproducibility.

Multi-color RGB marking enables clonality assessment of liver tumors in a murine xenograft model.

We recently introduced red-green-blue (RGB) marking for clonal cell tracking based on individual color-coding. Here, we applied RGB marking to study clonal development of liver tumors. Immortalized, non-tumorigenic human fetal hepatocytes expressing the human telomerase reverse transcriptase (FH-hTERT) were RGB-marked by simultaneous transduction with lentiviral vectors encoding mCherry, Venus, and Cerulean. Multi-color fluorescence microscopy was used to analyze growth characteristics of RGB-marked FH-hTERT in vitro and in vivo after transplantation into livers of immunodeficient mice with endogenous liver damage (uPA/SCID). After initially polyclonal engraftment we observed oligoclonal regenerative nodules derived from transplanted RGB-marked FH-hTERT. Some mice developed monochromatic invasive liver tumors; their clonal origin was confirmed both on the molecular level, based on specific lentiviral-vector insertion sites, and by serial transplantation of one tumor. Vector insertions in proximity to the proto-oncogene MCF2 and the transcription factor MITF resulted in strong upregulation of mRNA expression in the respective tumors. Notably, upregulated MCF2 and MITF expression was also observed in 21% and 33% of 24 human hepatocellular carcinomas analyzed. In conclusion, liver repopulation with RGB-marked FH-hTERT is a useful tool to study clonal progression of liver tumors caused by insertional mutagenesis in vivo and will help identifying genes involved in liver cancer.

Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent.

Hematopoietic stem cell (HSC)-based gene therapy trials are now moving toward the use of lentiviral vectors (LVs) with success. However, one challenge in the field remains: efficient transduction of HSCs without compromising their stem cell potential. Here we showed that measles virus glycoprotein-displaying LVs (hemagglutinin and fusion protein LVs [H/F-LVs]) were capable of transducing 100% of early-acting cytokine-stimulated human CD34+ (hCD34+) progenitor cells upon a single application. Strikingly, these H/F-LVs also allowed transduction of up to 70% of nonstimulated quiescent hCD34+ cells, whereas conventional vesicular stomatitis virus G (VSV-G)-LVs reached 5% at the most with H/F-LV entry occurring exclusively through the CD46 complement receptor. Importantly, reconstitution of NOD/SCIDγc-/- (NSG) mice with H/F-LV transduced prestimulated or resting hCD34+ cells confirmed these high transduction levels in all myeloid and lymphoid lineages. Remarkably, for resting CD34+ cells, secondary recipients exhibited increasing transduction levels of up to 100%, emphasizing that H/F-LVs efficiently gene-marked HSCs in the resting state. Because H/F-LVs promoted ex vivo gene modification of minimally manipulated CD34+ progenitors that maintained stemness, we assessed their applicability in Fanconi anemia, a bone marrow (BM) failure with chromosomal fragility. Notably, only H/F-LVs efficiently gene-corrected minimally stimulated hCD34+ cells in unfractionated BM from these patients. These H/F-LVs improved HSC gene delivery in the absence of cytokine stimulation while maintaining their stem cell potential. Thus, H/F-LVs will facilitate future clinical applications requiring HSC gene modification, including BM failure syndromes, for which treatment has been very challenging up to now.

Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles.

The biological relevance of extracellular vesicles (EV) in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC) also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced Arabidopsis thaliana-DNA (A.t.-DNA) as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR) techniques we detected high copy numbers of A.t.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified A.t.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of A.t.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development.