Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data.

Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n = 12) under different (patho)physiological conditions as well as in human-derived biofluids (n = 3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications.

Glioblastoma-Associated Mesenchymal Stem/Stromal Cells and Cancer-Associated Fibroblasts: Partners in Crime?

Tumor-associated mesenchymal stem/stromal cells (TA-MSCs) have been recognized as attractive therapeutic targets in several cancer types, due to their ability to enhance tumor growth and angiogenesis and their contribution to an immunosuppressive tumor microenvironment (TME). In glioblastoma (GB), mesenchymal stem cells (MSCs) seem to be recruited to the tumor site, where they differentiate into glioblastoma-associated mesenchymal stem/stromal cells (GA-MSCs) under the influence of tumor cells and the TME. GA-MSCs are reported to exert important protumoral functions, such as promoting tumor growth and invasion, increasing angiogenesis, stimulating glioblastoma stem cell (GSC) proliferation and stemness, mediating resistance to therapy and contributing to an immunosuppressive TME. Moreover, they could act as precursor cells for cancer-associated fibroblasts (CAFs), which have recently been identified in GB. In this review, we provide an overview of the different functions exerted by GA-MSCs and CAFs and the current knowledge on the relationship between these cell types. Increasing our understanding of the interactions and signaling pathways in relevant models might contribute to future regimens targeting GA-MSCs and GB-associated CAFs to inhibit tumor growth and render the TME less immunosuppressive.

Generation of post-surgical minimal residual disease models to investigate metastasis in soft tissue sarcoma patient-derived orthotopic xenografts.

Despite optimal multimodal treatment including surgical resection, 50%-80% of high-grade soft tissue sarcoma (STS) patients metastasize. Here, we present a protocol for the generation and use of post-surgical minimal residual disease models to investigate metastatic relapse in STS patient-derived xenografts. We describe steps for orthotopic engraftment of high-grade STS patient-derived tumor tissue. We then detail procedures for primary tumor resection with broad, negative resection margins and follow-up until metastases using MRI. For complete details on the use and execution of this protocol, please refer to Fischer et al. (2023).1.

DIRAS3 induces autophagy and enhances sensitivity to anti-autophagic therapy in KRAS-driven pancreatic and ovarian carcinomas.

Pancreatic ductal adenocarcinoma (PDAC) and low-grade ovarian cancer (LGSOC) are characterized by the prevalence of KRAS oncogene mutations. DIRAS3 is the first endogenous non-RAS protein that heterodimerizes with RAS, disrupts RAS clustering, blocks RAS signaling, and inhibits cancer cell growth. Here, we found that DIRAS3-mediated KRAS inhibition induces ROS-mediated apoptosis in PDAC and LGSOC cells with KRAS mutations, but not in cells with wild-type KRAS, by downregulating NFE2L2/Nrf2 transcription, reducing antioxidants, and inducing oxidative stress. DIRAS3 also induces cytoprotective macroautophagy/autophagy that may protect mutant KRAS cancer cells from oxidative stress, by inhibiting mutant KRAS, activating the STK11/LKB1-PRKAA/AMPK pathway, increasing lysosomal CDKN1B/p27 localization, and inducing autophagic gene expression. Treatment with chloroquine or the novel dimeric chloroquine analog DC661 significantly enhances DIRAS3-mediated inhibition of mutant KRAS tumor cell growth in vitro and in vivo. Taken together, our study demonstrates that DIRAS3 plays a critical role in regulating mutant KRAS-driven oncogenesis in PDAC and LGSOC.Abbreviations: AFR: autophagic flux reporter; ATG: autophagy related; CQ: chloroquine; DCFDA: 2'-7'-dichlorodihydrofluorescein diacetate; DIRAS3: DIRAS family GTPase 3; DOX: doxycycline; KRAS: KRAS proto-oncogene, LGSOC: low-grade serous ovarian cancer; MiT/TFE: microphthalmia family of transcription factors; NAC: N-acetylcysteine; PDAC: pancreatic ductal adenocarcinoma; ROS: reactive oxygen species; TFEB: transcription factor EB.

Histologic tumor type as a determinant of survival in hormone receptor-positive, HER2-negative, pT1-3 invasive ductal and lobular breast cancer.

PURPOSE: The aim of the study was to compare the difference in survival between invasive ductal (IDC) and lobular carcinoma (ILC). METHODS: Data of patients (n = 1843) with a hormone receptor-positive, HER2-negative, pT1-3 IDC or ILC cancer without distant metastasis, treated at the Ghent University Hospital over the time period 2001-2015, were analyzed. RESULTS: ILC represented 13.9% of the tumors, had a higher percentage of pT3 and pN3 stages than IDC, lymphovascular space invasion (LVSI) was less present and Ki-67 was mostly low. 73.9% of ILCs were grade 2, whereas IDC had more grade 1 and grade 3 tumors. Kaplan-Meier curves and log-rank testing showed a significant worse DFS for ILC with pN ≥ 1 than for their IDC counterpart. In a multivariable Cox regression analysis the histologic tumor type, ductal or lobular, was a determinant of DFS over 120 months (IDC as reference; hazard ratio for ILC 1.77, 95% CI 1.08-2.90) just as the ER Allred score (hazard ratio 0.84, 95% CI 0.78-0.91), LVSI (hazard ratio 1.75, 95% CI 1.12-2.74) and pN3 (hazard ratio 2.29, 95% CI 1.03-5.09). Determinants of OS over ten years were age (hazard ratio 1.05, 95% CI 1.02-1.07), LVSI (hazard ratio 3.62, 95% CI 1.92-6.82) and the ER Allred score (hazard ratio 0.80, 95% CI 0.73-0.89). CONCLUSION: The histologic tumor type, ductal or lobular, determines DFS in hormone receptor-positive, HER2-negative, pT1-3 breast cancer besides the ER Allred score, LVSI and pN3.

A preclinical platform for assessing long-term drug efficacy exploiting mechanically tunable scaffolds colonized by a three-dimensional tumor microenvironment.

BACKGROUND: Long-term drug evaluation heavily relies upon rodent models. Drug discovery methods to reduce animal models in oncology may include three-dimensional (3D) cellular systems that take into account tumor microenvironment (TME) cell types and biomechanical properties. METHODS: In this study we reconstructed a 3D tumor using an elastic polymer (acrylate-endcapped urethane-based poly(ethylene glycol) (AUPPEG)) with clinical relevant stiffness. Single cell suspensions from low-grade serous ovarian cancer (LGSOC) patient-derived early passage cultures of cancer cells and cancer-associated fibroblasts (CAF) embedded in a collagen gel were introduced to the AUPPEG scaffold. After self-organization in to a 3D tumor, this model was evaluated by a long-term (> 40 days) exposure to a drug combination of MEK and HSP90 inhibitors. The drug-response results from this long-term in vitro model are compared with drug responses in an orthotopic LGSOC xenograft mouse model. RESULTS: The in vitro 3D scaffold LGSOC model mimics the growth ratio and spatial organization of the LGSOC. The AUPPEG scaffold approach allows to test new targeted treatments and monitor long-term drug responses. The results correlate with those of the orthotopic LGSOC xenograft mouse model. CONCLUSIONS: The mechanically-tunable scaffolds colonized by a three-dimensional LGSOC allow long-term drug evaluation and can be considered as a valid alternative to reduce, replace and refine animal models in drug discovery.

Repositioning the antihistamine ebastine as an intracellular siRNA delivery enhancer.

Small interfering RNAs (siRNAs) are promising therapeutics for the treatment of human diseases via the induction of sequence-specific gene silencing. To be functional, siRNAs require cytosolic delivery into target cells. However, state-of-the-art delivery systems mediate cellular entry through endocytosis and suffer from ineffective endosomal escape, routing a substantial fraction of the siRNA towards the lysosomal compartment. Cationic amphiphilic drugs (CADs) have been described to improve cytosolic siRNA delivery by the transient induction of lysosomal membrane permeabilization. In this work, we evaluated ebastine, an antihistamine CAD, for its ability to enhance cytosolic release of siRNA in a non-small cell lung cancer model. In particular, we demonstrated that ebastine can improve the siRNA-mediated gene silencing efficiency of a polymeric nanogel by 40-fold, outperforming other CAD compounds. Additionally, ebastine substantially enhanced gene knockdown of a cholesterol-conjugated siRNA, in two-dimensional (2D) cell culture as well as in three-dimensional (3D) tumor spheroids. Finally, ebastine could strongly promote siRNA delivery of lipid nanoparticles (LNPs) composed of a pH-dependent switchable ionizable lipid and with stable PEGylation, in contrast to state-of-the-art LNP formulations. Altogether, we identified ebastine as a potent and versatile siRNA delivery enhancer in cancer cells, which offers opportunities for drug combination therapy in oncology.

Depletion of soluble cytokines unlocks the immunomodulatory bioactivity of extracellular vesicles.

Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration.

Endoglin, a Novel Biomarker and Therapeutical Target to Prevent Malignant Peripheral Nerve Sheath Tumor Growth and Metastasis.

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft-tissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGFß coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs. EXPERIMENTAL DESIGN: ENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathway activation and in vivo MPNST growth and metastasis, were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models. RESULTS: ENG expression was found to be upregulated in both human MPNST tumor tissues and plasma-circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Moreover, combination of anti-ENG therapy with MEK inhibition effectively reduced tumor cell growth and angiogenesis. CONCLUSIONS: Our data unveil a tumor-promoting function of ENG in MPNSTs and support the use of this protein as a novel biomarker and a promising therapeutic target for this disease.

Vascularized adipose tissue engineering: moving towards soft tissue reconstruction.

Soft tissue defects are a common clinical challenge mostly caused by trauma, congenital anomalies and oncological surgery. Current soft tissue reconstruction options include synthetic materials (fillers and implants) and autologous adipose tissue transplantation through flap surgery and/or lipotransfer. Both reconstructive options hold important disadvantages to which vascularized adipose tissue engineering (VATE) strategies could offer solutions. In this review, we first summarized pivotal characteristics of functional adipose tissue such as the structure, function, cell types, development and extracellular matrix (ECM). Next, we discussed relevant cell sources and how they are applied in different state-of-the-art VATE techniques. Herein, biomaterial scaffolds and hydrogels, ECMs, spheroids, organoids, cell sheets, three dimensional printing and microfluidics are overviewed. Also, we included extracellular vesicles and emphasized their potential role in VATE. Lastly, current challenges and future perspectives in VATE are pointed out to help to pave the road towards clinical applications.

Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility.

BACKGROUND: Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. RESULTS: Automated versus manual density-based separation of trackable recombinant extracellular vesicles (rEV) spiked in PBS significantly reduces variability in rEV recovery as quantified by fluorescent nanoparticle tracking analysis and ELISA. To validate automated density-based EV separation from complex body fluids, including blood plasma and urine, we assess reproducibility, recovery, and specificity by mass spectrometry-based proteomics and transmission electron microscopy. Method reproducibility is the highest in the automated procedure independent of the matrix used. While retaining (in urine) or enhancing (in plasma) EV recovery compared to manual liquid handling, automation significantly reduces the presence of body fluid specific abundant proteins in EV preparations, including apolipoproteins in plasma and Tamm-Horsfall protein in urine. CONCLUSIONS: In conclusion, automated liquid handling ensures cost-effective EV separation from human body fluids with high reproducibility, specificity, and reduced hands-on time with the potential to enable larger-scale biomarker studies.

Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics.

The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT; six preservation and five non-preservation) and three blood processing intervals (BPI; 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies.

Direct lysis of 3D cell cultures for RT-qPCR gene expression quantification.

In vitro cell culture experiments are widely used to study cellular behavior in most biological research fields. Except for suspension cells, most human cell types are cultured as adherent monolayers on a plastic surface. While technically convenient, monolayer cultures can suffer from limitations in terms of physiological relevance, as their resemblance to complex in vivo tissue structures is limited. To address these limitations, three-dimensional (3D) cell culture systems have gained increased interest as they mimic key structural and functional properties of their in vivo tissue counterparts. Nevertheless, protocols established on monolayer cell cultures may require adjustments if they are to be applied to 3D cell cultures. As gene expression quantification is an essential part of many in vitro experiments, we evaluated and optimized a direct cell lysis, reverse transcription and qPCR protocol applicable for 3D cell cultures. The newly developed protocol wherein gene expression is determined directly from crude cell lysates showed improved cell lysis compared to the standard protocol, accurate gene expression quantification, hereby avoiding time-consuming cell harvesting and RNA extraction.

Pembrolizumab, radiotherapy, and an immunomodulatory five-drug cocktail in pretreated patients with persistent, recurrent, or metastatic cervical or endometrial carcinoma: Results of the phase II PRIMMO study.

A phase II study (PRIMMO) of patients with pretreated persistent/recurrent/metastatic cervical or endometrial cancer is presented. Patients received an immunomodulatory five-drug cocktail (IDC) consisting of low-dose cyclophosphamide, aspirin, lansoprazole, vitamin D, and curcumin starting 2 weeks before radioimmunotherapy. Pembrolizumab was administered three-weekly from day 15 onwards; one of the tumor lesions was irradiated (8Gyx3) on days 15, 17, and 19. The primary endpoint was the objective response rate per immune-related response criteria (irORR) at week 26 (a lower bound of the 90% confidence interval [CI] of > 10% was considered efficacious). The prespecified 43 patients (cervical, n = 18; endometrial, n = 25) were enrolled. The irORR was 11.1% (90% CI 2.0-31.0) in cervical cancer and 12.0% (90% CI 3.4-28.2) in endometrial cancer. Median duration of response was not reached in both cohorts. Median interval-censored progression-free survival was 4.1 weeks (95% CI 4.1-25.7) in cervical cancer and 3.6 weeks (95% CI 3.6-15.4) in endometrial cancer; median overall survival was 39.6 weeks (95% CI 15.0-67.0) and 37.4 weeks (95% CI 19.0-50.3), respectively. Grade ≥ 3 treatment-related adverse events were reported in 10 (55.6%) cervical cancer patients and 9 (36.0%) endometrial cancer patients. Health-related quality of life was generally stable over time. Responders had a significantly higher proportion of peripheral T cells when compared to nonresponders (p = 0.013). In conclusion, PRIMMO did not meet its primary objective in both cohorts; pembrolizumab, radiotherapy, and an IDC had modest but durable antitumor activity with acceptable but not negligible toxicity.Trial registration ClinicalTrials.gov (identifier NCT03192059) and EudraCT Registry (number 2016-001569-97).

One cisplatin dose provides durable stimulation of anti-tumor immunity and alleviates anti-PD-1 resistance in an intraductal model for triple-negative breast cancer.

Aggressive triple-negative breast cancer (TNBC) is classically treated with chemotherapy. Besides direct tumor cell killing, some chemotherapeutics such as cisplatin provide additional disease reduction through stimulation of anti-tumor immunity. The cisplatin-induced immunomodulation in TNBC was here investigated in-depth using immunocompetent intraductal mouse models. Upon primary tumor transition to invasive carcinoma, cisplatin was injected systemically and significantly reduced tumor progression. Flow cytometric immunophenotyping was corroborated by immunohistochemical analyses and revealed both differential immune cell compositions and positivity for their programmed death (PD)-1 and PD-ligand (L)1 markers across body compartments, including the primary tumor, axillary lymph nodes and spleen. As key findings, a significant decrease in immunosuppressive and a concomitant increase in anti-tumor lymphocytic cell numbers were observed in the axillary lymph nodes and spleen, highlighting their importance in cisplatin-stimulated anti-tumor immunity. These immunomodulatory effects were already established following the first cisplatin dose, indicating that early cisplatin-mediated events may determine (immuno)therapeutic outcome. Furthermore, a single cisplatin dose sufficed to alleviate anti-PD-1 resistance in a 4T1-based model, providing add-on disease reduction without toxic side effects as seen upon multiple cisplatin dosing. Overall, these results highlight cisplatin as immunotherapeutic ally in TNBC, providing durable immunostimulation, even after a single dose.

The quorum sensing peptide EntF* promotes colorectal cancer metastasis in mice: a new factor in the host-microbiome interaction.

BACKGROUND: Colorectal cancer, one of the most common malignancies worldwide, is associated with a high mortality rate, mainly caused by metastasis. Comparative metagenome-wide association analyses of healthy individuals and cancer patients suggest a role for the human intestinal microbiota in tumor progression. However, the microbial molecules involved in host-microbe communication are largely unknown, with current studies mainly focusing on short-chain fatty acids and amino acid metabolites as potential mediators. Quorum sensing peptides are not yet considered in this context since their presence in vivo and their ability to affect host cells have not been reported so far. RESULTS: Here, we show that EntF*, a metabolite of the quorum sensing peptide EntF produced by Enterococcus faecium, is naturally present in mice bloodstream. Moreover, by using an orthotopic mouse model, we show that EntF* promotes colorectal cancer metastasis in vivo, with metastatic lesions in liver and lung tissues. In vitro tests suggest that EntF* regulates E-cadherin expression and consequently the epithelial-mesenchymal transition, via the CXCR4 receptor. In addition, alanine-scanning analysis indicates that the first, second, sixth, and tenth amino acid of EntF* are critical for epithelial-mesenchymal transition and tumor metastasis. CONCLUSION: Our work identifies a new class of molecules, quorum sensing peptides, as potential regulators of host-microbe interactions. We prove, for the first time, the presence of a selected quorum sensing peptide metabolite in a mouse model, and we demonstrate its effects on colorectal cancer metastasis. We believe that our work represents a starting point for future investigations on the role of microbiome in colorectal cancer metastasis and for the development of novel bio-therapeutics in other disease areas.

Epithelial to mesenchymal transition influences fibroblast phenotype in colorectal cancer by altering miR-200 levels in extracellular vesicles.

Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer-associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell-cell communication. Epithelial CRC EVs suppressed TGF-ß-driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR-200 (miR-200a/b/c, -141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR-200 expression or ZEB1 knockdown, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR-200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co-injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR-200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV-mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF rich.

Heterogeneity of CAFfeinated Tumors: Sweet Targeting Perspectives.

The tumor microenvironment (TME) shows heterogeneity within a tumor. An important element of the TME is cancer-associated fibroblasts (CAF). In this issue of Cancer Research, Bouchard and colleagues investigate the heterogeneity of CAFs from spatially different zones of lung adenocarcinoma resection specimens. Multiomics analysis revealed changes in the O-glycoproteome, unique to CAFs from the tumor edge, an effect reinforced by contact with cancer cells. This O-glycoprotein signature offers unique targeting perspectives that reciprocally affect cancer cell epithelial-to-mesenchymal plasticity, a key mechanism of cancer progression. Deeper understanding of the cancer-stimulating and cancer-inhibiting role of CAF subtypes will facilitate the development of CAF-directed therapeutic approaches. See related article by Bouchard et al., p. 648.

Systemically circulating bacterial extracellular vesicles: origin, fate, and function.

Bacteria contribute to human host (patho)physiology through the production of a myriad of biomolecules enclosed in membrane vesicles [bacterial extracellular vesicles (BEVs)]. Recent research revealed that BEVs, as a functional output of bacteria, enter the systemic circulation. Here, we highlight the current state of knowledge on the origin, translocation, distribution, function, and excretion or elimination of systemically circulating BEVs and delineate knowledge gaps. Further investigations on the so far occult stages of BEV entry beyond the walls of epithelial and immune barriers will unmask the role of BEVs in health and disease.