Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo ...to recipient cells to affect the stability of individual mRNAs and the cells' transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we defined multiple properties of EVs and analyzed their capacity to deliver packaged miRNAs into target cells to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but EVs did not fuse detectably with cellular membranes to deliver their cargo. We engineered EVs to be fusogenic and document their capacity to deliver functional messenger RNAs. Engineered fusogenic EVs, however, did not detectably alter the functionality of cells exposed to miRNA-carrying EVs. These results suggest that EV-borne miRNAs do not act as effectors of cell-to-cell communication.
Lifelong persistence of Epstein-Barr virus (EBV) in infected hosts is mainly owed to the virus' pronounced abilities to evade immune responses of its human host. Active immune evasion mechanisms ...reduce the immunogenicity of infected cells and are known to be of major importance during lytic infection. The EBV genes BCRF1 and BNLF2a encode the viral homologue of IL-10 (vIL-10) and an inhibitor of the transporter associated with antigen processing (TAP), respectively. Both are known immunoevasins in EBV's lytic phase. Here we describe that BCRF1 and BNLF2a are functionally expressed instantly upon infection of primary B cells. Using EBV mutants deficient in BCRF1 and BNLF2a, we show that both factors contribute to evading EBV-specific immune responses during the earliest phase of infection. vIL-10 impairs NK cell mediated killing of infected B cells, interferes with CD4+ T-cell activity, and modulates cytokine responses, while BNLF2a reduces antigen presentation and recognition of newly infected cells by EBV-specific CD8+ T cells. Together, both factors significantly diminish the immunogenicity of EBV-infected cells during the initial, pre-latent phase of infection and may improve the establishment of a latent EBV infection in vivo.
Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer ...metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage.
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•DeepMACT is a deep learning-based pipeline for comprehensive analysis of metastases•DeepMACT identifies micrometastases and single cancer cells in full-body 3D scans•DeepMACT reveals the efficacy of antibody-drug targeting in the entire body•DeepMACT indicates that the tumor microenvironment affects drug targeting efficacy
Deep learning-based automated detection and quantification of micrometastases and therapeutic antibody targeting down to the level of single disseminated cancer cells provides unbiased analysis of multiple metastatic cancer models at the full-body scale.
Carbonic anhydrase XII (CAXII) is a membrane‐tethered ectoenzyme involved in intracellular pH regulation and overexpressed across various types of human cancer. Because CAXII inhibition shows ...antitumor activity in vitro, it is thought that the enzyme is mandatory for maximum tumor growth, above all under hypoxic conditions. Recently, it has been shown that CAXII is co‐expressed along with the P‐glycoprotein (P‐GP) on many tumor cells and that both proteins physically interact. Of interest, blocking CAXII activity also decreases P‐GP activity in cancer cells both in vitro and in vivo. Previously, we have reported on the development of a monoclonal antibody, termed 6A10, which specifically and efficiently blocks human CAXII activity. Here, we demonstrate that 6A10 also indirectly reduces P‐GP activity in CAXII/P‐GP double‐positive chemoresistant cancer cells, resulting in enhanced chemosensitivity as revealed by enhanced accumulation of anthracyclines and increased cell death in vitro. Even more important, we show that mice carrying human triple‐negative breast cancer xenografts co‐treated with doxorubicin (DOX) and 6A10 show a significantly reduced number of metastases. Collectively, our data provide evidence that the inhibition of CAXII with 6A10 is an attractive way to reduce chemoresistance of cancer cells and to interfere with the metastatic process in a clinical setting.
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Through the reversible hydratation of carbon dioxide, carbonic anhydrases (CAs) produce bicarbonate, a critical mediator of pH homeostasis. In human cancers, however, certain forms of CA are overexpressed and may provide a survival advantage for hypoxic tumor cells in acidic tumor environments. Here, using a monoclonal antibody, 6A10, the authors show that inhibition of cancer‐associated CAXII enhances chemosensitivity in resistant cancer cells. In an orthotopic breast cancer mouse model, co‐treatment with 6A10 and doxorubicin had no inhibitory effect on primary tumor growth. In tumor‐bearing animals, however, co‐treatment with the drugs significantly reduced the number of lung metastases.
The common pathogen Epstein-Barr virus (EBV) transforms normal human B cells and can cause cancer. Latent membrane protein 2A (LMP2A) of EBV supports activation and proliferation of infected B cells ...and is expressed in many types of EBV-associated cancer. It is not clear how latent EBV infection and cancer escape elimination by host immunity, and it is unknown whether LMP2A can influence the interaction of EBV-infected cells with the immune system. We infected primary B cells with EBV deleted for LMP2A, and established lymphoblastoid cell lines (LCLs). We found that CD8+ T cell clones showed higher reactivity against LMP2A-deficient LCLs compared to LCLs infected with complete EBV. We identified several potential mediators of this immunomodulatory effect. In the absence of LMP2A, expression of some EBV latent antigens was elevated, and cell surface expression of MHC class I was marginally increased. LMP2A-deficient LCLs produced lower amounts of IL-10, although this did not directly affect CD8+ T cell recognition. Deletion of LMP2A led to several changes in the cell surface immunophenotype of LCLs. Specifically, the agonistic NKG2D ligands MICA and ULBP4 were increased. Blocking experiments showed that NKG2D activation contributed to LCL recognition by CD8+ T cell clones. Our results demonstrate that LMP2A reduces the reactivity of CD8+ T cells against EBV-infected cells, and we identify several relevant mechanisms.
Herpesviruses are dsDNA viruses, but their virions may additionally contain RNAs that can be transduced to recipient cells. The biological functions of herpes virion RNA species are unknown. Here we ...address this issue for EBV, a widespread human herpesvirus with oncogenic potential. We show that EBV-derived particles that include virions, virus-like particles, and subviral vesicles contain viral mRNAs, microRNAs, and other noncoding RNAs. Viral RNAs were transduced during infection and deployed immediate functions that enhanced EBV’s capacity to transform primary B cells. Among these transduced viral RNAs, BZLF1 transcripts transactivated viral promoters triggering the prelatent phase of EBV infection, noncoding EBV-encoded RNA transcripts induced cellular cytokine synthesis, and BNLF2a mRNA led to immune evasion that prevented T-cell responses to newly infected B cells. Hence, transduced viral RNAs govern critical processes immediately after infection of B cells with EBV and likely play important roles in herpesviral infection in general.
Epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein expressed on adenomatous and simple epithelia, where it is involved in homophilic adhesion at the basolateral membrane. Carcinomas ...strongly overexpress EpCAM through an, as yet, unknown mechanism. Interestingly, otherwise EpCAM-negative squamous epithelia are seen to express EpCAM concomitant with their transformation and de-differentiation. The amount of EpCAM and the number of expressing cells both increase with the grade of dysplasia. Despite an important amount of data correlating the expression of EpCAM with cellular proliferation and de-differentiation, such as the coexpression with Ki-67, a marker for proliferation, it is unknown whether EpCAM may directly contribute to carcinogenesis. Here, we show that EpCAM has a direct impact on cell cycle and proliferation, and the ability to rapidly upregulate the proto-oncogene c-myc and cyclin A/E. Human epithelial 293 cells as well as murine NIH3T3 fibroblasts expressing EpCAM had a decreased requirement for growth factors, enhanced metabolic activity and colony formation capacity. Importantly, the inhibition of EpCAM expression with antisense mRNA led to a strong decrease in proliferation and metabolism in human carcinoma cells. Moreover, domain swapping experiments demonstrated that the intracellular part of EpCAM is necessary and sufficient to transduce the effects described. Thus, the data presented here highlight the role of EpCAM, demonstrating for the first time a direct link to cell cycle and proliferation.
We report for the first time Antibody-Drug-Conjugates (ADCs) containing human (h) Carbonic Anhydrase (CA; EC 4.2.1.1) directed Monoclonal Antibodies (MAbs) linked to low molecular weight inhibitors ...of the same enzymes by means of hydrophilic peptide spacers. In agreement with the incorporated CA directed MAb fragments,
inhibition data of the obtained ADCs showed sub-nanomolar K
values for the tumour associated CAs IX and XII which were up to 10-fold more potent when compared to the corresponding unconjugated MAbs. In addition, the introduction of the CA inhibitor (CAI) benzenesulfonamide allowed the ADCs to potently inhibit the housekeeping tumoral off-target human CA II isoform. Such results are supporting the definition of an unprecedented reported class of ADCs able to hit simultaneously multiple hCAs physiologically cooperative in maintaining altered cellular metabolic pathways, and therefore ideal for the treatment of chronic diseases such as cancers and inflammation diseases.
Targeting the tumor-associated carbonic anhydrase XII (CA XII) is considered a promising strategy to improve cancer treatment. As such progress is highly demanded for ovarian carcinomas, the present ...study aimed to provide deeper information about their CA XII expression profile. A large collection of tissue specimens was stained immunohistochemically with a specific anti-CA XII antibody to evaluate the expression in neoplastic and non-neoplastic epithelial ovarian cells. In addition, flow cytometry was used to measure CA XII expression on tumor cells from malignant ascites fluid. Binding of the antibody revealed a significant CA XII expression in most ovarian carcinoma tissue samples and ascites-derived ovarian carcinoma cells. Moreover, CA XII was expressed at higher levels in ovarian carcinomas as compared to borderline ovarian tumors and non-neoplastic ovarian epithelia. Within the carcinoma tissues, high expression of CA XII was associated with higher tumor grading and a trend towards shorter overall survival. Our results indicate that CA XII plays a crucial role for the malignancy of ovarian carcinoma cells and emphasize the potential of CA XII as a diagnostic marker and therapeutic target in the management of ovarian carcinomas.
gp350, the major envelope protein of Epstein-Barr-Virus, confers B-cell tropism to the virus by interacting with the B lineage marker CD21. Here we utilize gp350 to generate tailored exosomes with an ...identical tropism. These exosomes can be used for the targeted co-transfer of functional proteins to normal and malignant human B cells. We demonstrate here the co-transfer of functional CD154 protein on tailored gp350+ exosomes to malignant B blasts from patients with B chronic lymphocytic leukemia (B-CLL), rendering B blasts immunogenic to tumor-reactive autologous T cells. Intriguingly, engulfment of gp350+ exosomes by B-CLL cells and presentation of gp350-derived peptides also re-stimulated EBV-specific T cells and redirected the strong antiviral cellular immune response in patients to leukemic B cells. In essence, we show that gp350 alone confers B-cell tropism to exosomes and that these exosomes can be further engineered to simultaneously trigger virus- and tumor-specific immune responses. The simultaneous exploitation of gp350 as a tropism molecule for tailored exosomes and as a neo-antigen in malignant B cells provides a novel attractive strategy for immunotherapy of B-CLL and other B-cell malignancies.
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