Extracellular vesicle-mediated transfer of genetic information between the hematopoietic system and the brain in response to inflammation.

Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.

Body fluid exosomes promote secretion of inflammatory cytokines in monocytic cells via Toll-like receptor signaling.

Tumor-derived exosomes have been shown to induce various immunomodulatory effects. However, the underlying signaling pathways are poorly understood. Here, we analyzed the effects of ex vivo-derived exosomes on monocytic cell differentiation/activation using THP-1 cells as model. We isolated exosomes from various body fluids such as amniotic fluid, liver cirrhosis ascites, and malignant ascites of ovarian cancer patients. We observed that exosomes were internalized by THP-1 cells and induced the production of IL-1ß, TNF-α, and IL-6. Analysis of the signaling pathways revealed a fast triggering of NFκB and a delayed activation of STAT3. Pharmacologic and antibody-blocking experiments showed that the initial production of IL-6 was instrumental for subsequent activation of STAT3. Importantly, triggering of cell signaling was not a unique property of tumor exosomes but was also observed with exosomes of noncancerous origin. Exosomal signaling was TLR-dependent as the knockdown of Toll-like receptor 2 (TLR2) and TLR4 blocked NFκB and STAT3 activation. Similar results were obtained with TLR-neutralizing antibodies. Exosomes also triggered the release of cytokines from mouse bone marrow-derived dendritic cells or macrophages. This process was MyD88-dependent, further supporting a role of TLR signaling. Our results suggest that exosomes trigger TLR-dependent signaling pathways in monocytic precursor cells but possibly also in other immune cells. This process could be important for the induction of immunosuppressive mechanisms during cancer progression and inflammatory diseases.

INTERCEPT H3: a multicenter phase I peptide vaccine trial for the treatment of H3-mutated diffuse midline gliomas.

INTRODUCTION: Diffuse midline gliomas (DMG) are universally lethal central nervous system tumors that carry almost unanimously the clonal driver mutation histone-3 K27M (H3K27M). The single amino acid substitution of lysine to methionine harbors a neoantigen that is presented in tumor tissue. The long peptide vaccine H3K27M-vac targeting this major histocompatibility complex class II (MHC class II)-restricted neoantigen induces mutation-specific immune responses that suppress the growth of H3K27M+ flank tumors in an MHC-humanized rodent model. METHODS: INTERCEPT H3 is a non-controlled open label, single arm, multicenter national phase 1 trial to assess safety, tolerability and immunogenicity of H3K27M-vac in combination with standard radiotherapy and the immune checkpoint inhibitor atezolizumab (ATE). 15 adult patients with newly diagnosed K27M-mutant histone-3.1 (H3.1K27M) or histone-3.3 (H3.3K27M) DMG will be enrolled in this trial. The 27mer peptide vaccine H3K27M-vac will be administered concomitantly to standard radiotherapy (RT) followed by combinatorial treatment with the programmed death-ligand 1 (PD-L1) targeting antibody ATE. The first three vaccines will be administered bi-weekly (q2w) followed by a dose at the beginning of recovery after RT and six-weekly administrations of doses 5 to 11 thereafter. In a safety lead-in, the first three patients (pts. 1-3) will be enrolled sequentially. PERSPECTIVE: H3K27M-vac is a neoepitope targeting long peptide vaccine derived from the clonal driver mutation H3K27M in DMG. The INTERCEPT H3 trial aims at demonstrating (1) safety and (2) immunogenicity of repeated fixed dose vaccinations of H3K27M-vac administered with RT and ATE in adult patients with newly diagnosed H3K27M-mutant DMG. TRIAL REGISTRATION: NCT04808245.

CD24 Ala57Val polymorphism predicts pathologic complete response to sequential anthracycline- and taxane-based neoadjuvant chemotherapy for primary breast cancer.

Overexpression of CD24 is an independent prognostic factor for breast cancer. Recently, two polymorphisms in the CD24 gene were linked to disease risk and progression in autoimmune diseases. Here, we evaluated the clinical relevance of these polymorphisms with respect to their potential to predict a pathologic complete response (pCR) to neoadjuvant chemotherapy (NCT) for primary breast cancer (PBC), one of the strongest prognostic factors in this setting. A total of 257 patients were randomized to either doxorubicin/cyclophosphamide (AC) or doxorubicin/pemetrexed (AP), both followed by docetaxel (Doc) as NCT for T2-4 N0-2 M0 PBC as part of an international, multicenter, randomized phase II trial. CD24 polymorphisms were analyzed on germ line DNA and correlated with clinicopathologic variables and pCR. No significant associations were found between either of the polymorphisms and any of the clinicopathologic variables. In a multivariate analysis, CD24 Val/Val genotype was the only significant predictor of pCR (OR: 4.97; P = 0.003). The predictive potential was significant in both treatment arms and in the hormone receptor-positive subgroup. There was no correlation between CD24 3'UTR (TG/Del) genotype and pCR. We did not observe any association between CD24 genotype and CD24 protein expression or in vitro chemosensitivity, but there was a significant correlation between CD24 Val/Val and intratumoral lymphocyte aggregates. In conclusion, CD24 Ala/Val SNP is a strong and independent predictor of pCR after NCT for PBC and may affect immune functions rather than tumor characteristics. Further evaluation of the CD24 function and validation of its predictive potential are clearly warranted.

AMPLIFY-NEOVAC: a randomized, 3-arm multicenter phase I trial to assess safety, tolerability and immunogenicity of IDH1-vac combined with an immune checkpoint inhibitor targeting programmed death-ligand 1 in isocitrate dehydrogenase 1 mutant gliomas.

INTRODUCTION: Isocitrate dehydrogenase (IDH) mutations are disease-defining mutations in IDH-mutant astrocytomas and IDH-mutant and 1p/19q-codeleted oligodendrogliomas. In more than 80% of these tumors, point mutations in IDH type 1 (IDH1) lead to expression of the tumor-specific protein IDH1R132H. IDH1R132H harbors a major histocompatibility complex class II (MHCII)-restricted neoantigen that was safely and successfully targeted in a first-in human clinical phase 1 trial evaluating an IDH1R132H 20-mer peptide vaccine (IDH1-vac) in newly diagnosed astrocytomas concomitant to standard of care (SOC). METHODS: AMPLIFY-NEOVAC is a randomized, 3-arm, window-of-opportunity, multicenter national phase 1 trial to assess safety, tolerability and immunogenicity of IDH1-vac combined with avelumab (AVE), an immune checkpoint inhibitor (ICI) targeting programmed death-ligand 1 (PD-L1). The target population includes patients with resectable IDH1R132H-mutant recurrent astrocytoma or oligodendroglioma after SOC. Neoadjuvant and adjuvant immunotherapy will be administered to 48 evaluable patients. In arm 1, 12 patients will receive IDH1-vac; in arm 2, 12 patients will receive the combination of IDH1-vac and AVE, and in arm 3, 24 patients will receive AVE only. Until disease progression according to immunotherapy response assessment for neuro-oncology (iRANO) criteria, treatment will be administered over a period of maximum 43 weeks (primary treatment phase) followed by facultative maintenance treatment. PERSPECTIVE: IDH1R132H 20-mer peptide is a shared clonal driver mutation-derived neoepitope in diffuse gliomas. IDH1-vac safely targets IDH1R132H in newly diagnosed astrocytomas. AMPLIFY-NEOVAC aims at (1) demonstrating safety of enhanced peripheral IDH1-vac-induced T cell responses by combined therapy with AVE compared to IDH1-vac only and (2) investigating intra-glioma abundance and phenotypes of IDH1-vac induced T cells in exploratory post-treatment tissue analyses. In an exploratory analysis, both will be correlated with clinical outcome. TRIAL REGISTRATION: NCT03893903.

Body fluid derived exosomes as a novel template for clinical diagnostics.

BACKGROUND: Exosomes are small membrane vesicles with a size of 40-100 nm that are released by different cell types from a late endosomal cellular compartment. They can be found in various body fluids including plasma, malignant ascites, urine, amniotic fluid and saliva. Exosomes contain proteins, miRNAs and mRNAs (exosome shuttle RNA, esRNA) that could serve as novel platform for diagnosis. METHOD: We isolated exosomes from amniotic fluid, saliva and urine by differential centrifugation on sucrose gradients. Marker proteins were identified by Western blot and FACS analysis after adsorption of exosomes to latex beads. We extracted esRNA from exosomes, carried out RT-PCR, and analyzed amplified products by restriction length polymorphism. RESULTS: Exosomes were positive for the marker proteins CD24, CD9, Annexin-1 and Hsp70 and displayed the correct buoyant density and orientation of antigens. In sucrose gradients the exosomal fractions contained esRNA that could be isolated with sufficient quantity for further analysis. EsRNAs were protected in exosomes from enzymatic degradation. Amniotic fluid esRNA served as template for the typing of the CD24 single nucleotide polymorphism (rs52812045). It also allowed sex determination of the fetus based on the detection of the male specific ZFY gene product. CONCLUSIONS: Our data demonstrate that exosomes from body fluids carry esRNAs which can be analyzed and offers access to the transcriptome of the host organism. The exosomal lipid bilayer protects the genetic information from degradation. As the isolation of exosomes is a minimally invasive procedure, this technique opens new possibilities for diagnostics.

Loss of EpCAM expression in breast cancer derived serum exosomes: role of proteolytic cleavage.

OBJECTIVE: Cancer cells in the body release soluble and membranous factors that manipulate the tumor environment to facilitate growth and survival. Recent years have provided evidence that small microvesicles that are termed exosomes may play a pivotal role in this process. Exosomes are membrane vesicles with a size of 40-100 nm that are released by both tumor and normal cells and can be found in various body fluids. Tumor-derived exosomes carry functional proteins, mRNAs, and miRNAs and could serve as novel platform for tumor diagnosis and prognosis. However, marker proteins that allow enrichment of tumor-derived exosomes over normal exosomes are less well defined. METHODS: We used Western blot analysis and antibody coupled magnetic beads to characterize CD24 and EpCAM as markers for exosomes. We investigated ovarian carcinoma ascites, pleural effusions and serum of breast carcinoma patients. As non-tumor derived control we used exosomes from ascites of liver cirrhosis patients. RESULTS: Exosomes could be isolated from all body fluids and contained marker proteins as well as miRNAs. We observed that CD24 and EpCAM were selectively present on ascites exosomes of tumor patients and copurified together on anti-EpCAM or anti-CD24 magnetic beads. In breast cancer patients CD24 was present but EpCAM was absent from serum exosomes. Instead, the intact EpCAM ectodomain was recovered in a soluble form. We provide evidence that EpCAM can be cleaved from exosomes via serum metalloproteinase(s). CONCLUSION: Loss of EpCAM on serum exosomes may hamper enrichment by immune-affinity isolation. We suggest that CD24 could be an additional marker for the enrichment of tumor-derived exosomes from blood.

Extracellular vesicle-mediated transfer of functional RNA in the tumor microenvironment.

Extracellular vesicles (EVs) have been shown to transfer various molecules, including functional RNA between cells and this process has been suggested to be particularly relevant in tumor-host interactions. However, data on EV-mediated RNA transfer has been obtained primarily by in vitro experiments or involving ex vivo manipulations likely affecting its biology, leaving their physiological relevance unclear. We engineered glioma and carcinoma tumor cells to express Cre recombinase showing their release of EVs containing Cre mRNA in various EV subfractions including exosomes. Transplantation of these genetically modified tumor cells into mice with a Cre reporter background leads to frequent recombination events at the tumor site. In both tumor models the majority of recombined cells are CD45+ leukocytes, predominantly Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs). In addition, multiple lineages of recombined cells can be observed in the glioma model. In the lung carcinoma model, recombined MDSCs display an enhanced immunosuppressive phenotype and an altered miRNA profile compared to their non-recombined counterparts. Cre-lox based tracing of tumor EV RNA transfer in vivo can therefore be used to identify individual target cells in the tumor microenvironment for further mechanistical or functional analysis.

Role of miR-34a as a suppressor of L1CAM in endometrial carcinoma.

L1CAM promotes cell motility, invasion and metastasis formation in various human cancers and can be considered as a driver of tumor progression. Knowledge about genetic processes leading to the presence of L1CAM in cancers is of considerable importance. Experimentally, L1CAM expression can be achieved by various means. Over-expression of the transcription factor SLUG or treatment of cells with TGF-ß1 can induce or augment L1CAM levels in cancer cells. Likewise, hypomethylation of the L1CAM promoter on the X chromosome correlates with L1CAM expression. However, presently no mechanisms that might control transcriptional activity are known. Here we have identified miR-34a as a suppressor of L1CAM. We observed that L1CAM positive endometrial carcinoma (EC) cell lines HEC1B and SPAC1L lost L1CAM protein and mRNA by treatment with demethylating agents or knock-down of the DNA-methyltransferase-1 (DNMT1). Concomitantly, several miRNAs were up-regulated. Using miRNA profiling, luciferase reporter assays and mutagenesis, we identified miR-34a as a putative binder to the L1CAM-3'UTR. Over-expression of miR-34a in HEC1B cells blocked L1CAM expression and inhibited cell migration. In ECC1 cells (wildtype p53) the activation of p53 caused miR-34a up-regulation and loss of L1CAM expression that was miR-34a dependent. We observed an inverse correlation between L1CAM and miR-34a levels in EC cell lines. In primary tumor sections areas expressing high amounts of L1CAM had less miR-34a expression than those with low L1CAM levels. Our data suggest that miR-34a can regulate L1CAM expression by targeting L1CAM mRNA for degradation. These findings shed new light on the complex regulation of L1CAM in human tumors.

Exosomes as a potential tool for a specific delivery of functional molecules.

Extracellular membrane vesicles derived from the endosomal compartments and released by the fusion of the multivesicular bodies with the cell membrane are referred as exosomes (Exo) [Van Niel et al., J Biochem 140:13-21, 2006]. They function as mediators of intercellular communication and are employed by the organism in the regulation of systemic and local processes. Meantime, Exo are recognized as an indispensable entity of physiological fluids [Caby et al., Int Immunol 17:879-887, 2005; Lasser et al., J Transl Med 9:9, 2011; Lasser et al., Am J Rhinol Allergy 25:89-93, 2011]. Exo and other types of extracellular vesicles, e.g., exosome-like vesicles [van Niel et al., Gastroenterology 121:337-349, 2001] and microvesicles (MV) [Daveloose et al., Thromb Res 22:195-201, 1981], contain multiple functional molecules including lipids [Vidal et al., J Cell Physiol 140:455-462, 1989]; proteins [Simpson et al., Expert Rev Proteomics 6:267-283, 2009]; mRNA [Valadi et al., Nat Cell Biol 9:654-659, 2007]; DNA [Waldenstrom et al., PLoS One 7:e34653, 2012]; noncoding RNA, e.g., miRNA [Simpson et al., Expert Rev Proteomics 6:267-283, 2009]; and retrotransposon elements [Balaj et al., Nat Commun 2:180, 2011]. Assessment of the biological functions of Exo showed that they deliver specifically their cargo from the donor to recipient cells. Albeit the molecular mechanisms of this process are not fully understood, approaches for the application of Exo and MV as a tool for a cell-specific delivery of signalling molecules were successfully tested in in vitro and in vivo models [Maguire et al., Mol Ther 20:960-971, 2012]. Ovarian cancer cells release Exo, which bind stroma cells as well as donor cancer cells [Escrevente et al., BMC Cancer 11:108, 2011]. Here we describe an experimental approach for the assessment of Exo interaction and uptake by target cells. Methods for the isolation and purification of Exo from cell culture supernatants are included. To allow visualization of vesicle uptake, labelling of Exo with different fluorescent dyes, such as CFSE, PKH, DHPE, and DiOC18, is presented. Finally, we explain qualitative and quantitative analysis of Exo uptake by immunofluorescence and flow cytometry, respectively.