Virus infection can trigger extrinsic apoptosis. Cell-surface death receptors of the tumor necrosis factor family mediate this process. They either assist persistent viral infection or elicit the elimination of infected cells by the host. Death receptor-mediated apoptosis plays an important role in viral pathogenesis and the host antiviral response. Many viruses have acquired the capability to subvert death receptor-mediated apoptosis and evade the host immune response, mainly by virally encoded gene products that suppress death receptor-mediated apoptosis. In this review, we summarize the current information on virus infection and death receptor-mediated apoptosis, particularly focusing on the viral proteins that modulate death receptor-mediated apoptosis. Apoptosis, necroptosis, and pyroptosis are the three major ways of programed cell death (PCD) following virus infection ,2. Among2+ overload and endoplasmic reticulum stress. These heterogeneous apoptotic signals converge to trigger one pivotal event—mitochondrial outer membrane permeabilization (MOMP), which promotes the release of cytochrome c and other mitochondrial factors into the cytosol, ultimately leading to the generation of initiator and effector caspases and subsequent cell death [Apoptosis can be triggered by two distinct signaling pathways, namely the intrinsic and extrinsic pathways ,8,9. Thell death ,10. MOMPll death ,12. Actill death ,14. The ll death . Death rll death ,16,17. Lll death ,19. Unlill death . In typell death .L) and c-FLIP short (c-FLIPS) [Signaling through both Fas and TRAIL-R1/-R2 leads to the oligomerization of death receptors and intracellular assembly of DISC composing of dead receptor, the adaptor molecule FADD (FAS-associated with a death domain), procaspase-8, procaspase-10 and cellular FADD-like interleukin-1β converting enzyme (FLICE)-like inhibitory protein (c-FLIP). c-FLIP has two protein isoforms, c-FLIP long (c-FLIPc-FLIPS) . c-FLIP c-FLIPS) ,24,25. Ic-FLIPS) ,26.Death receptor-mediated apoptosis represents an efficient mechanism by which the virus can induce cell death and disseminate progeny, which plays an important role in viral pathogenesis and provides a potential therapeutic target. Regulation of death receptor-mediated apoptosis by the virus is mainly through upregulation of death receptors or their ligand on the cell surface of the infected individuals and increased sensitivity of the cells to death receptor-mediated apoptosis . Many viApoptosis mediated by death receptors plays an important role during HIV-1 infection. An increased expression of Fas antigen in CD4+ and CD8+ T lymphocytes occurs in patients infected with HIV-1, rendering those cells sensitive to FasL/Fas system-mediated apoptosis and contributing to T lymphocyte depletion in HIV-infected individuals ,104,105.Regulation of TRAIL expression and TRAIL-mediated apoptosis during HIV-1 infection has been well demonstrated. HIV-1 infection induces expression of TRAIL and DR5 and leads to TRAIL-mediated apoptosis in primary CD4+ T cells, which is regulated by IFN-α that is produced by HIV-1-stimulated plasmacytoid dendritic cells (pDCs) ,108. UprHIV-1-encoded proteins modulate death receptor-mediated apoptosis in different cell types. HIV-1 Tat, Vpu, gp120 and gp160 proteins sensitize T cells to Fas-mediated apoptosis with different regulatory mechanisms, possibly contributing to T-cell depletion in AIDS ,31,34. HIn chronic hepatitis C virus (HCV) infection, enhanced hepatocyte apoptosis and upregulation of the death receptors and death-inducing ligands have been described ,115,116.The pro- and anti-apoptotic roles of HCV proteins are controversial and dependent on the experimental system used . HCV corFas antigen expression significantly increases on PBMCs obtained from varicella-zoster virus (VZV) seropositive donors after culture with VZV antigen. The cultured cells undergo Fas-mediated apoptosis, suggesting a potential role of Fas-mediated apoptosis in the elimination of lymphocytes activated by VZV infection . AnotherInfluenza virus infection activates Fas gene expression and induces apoptosis of infected cells ,131,132.Human papillomavirus (HPV) E2 protein induces apoptosis mediated by FasL and TNF-α in HPV-positive and negative cervical cancer cell lines through interacting with c-FLIP and abrogating the apoptosis-inhibitory function of c-FLIP . HPV E7 Elimination of infected cells via death receptor-mediated apoptosis is one of the defense mechanisms against virus infection. Induction of early cell death would severely limit virus production and reduce or eliminate the spread of progeny virus in the host. Thus, many viruses have evolved many different strategies to interfere with death receptor signaling and prevent apoptosis through virally encoded antiapoptotic factors , therebyUL37 gene, inhibits Fas-mediated apoptosis at a point downstream of caspase-8 activation and Bid cleavage [Herpesviruses have been most instructive for viral inhibitors of death receptor-mediated apoptosis . Herpes cleavage . HCMV ULcleavage ,62. MCMVcleavage . Additiocleavage .Like poxvirus molluscum contagiosum virus (MCV)-encoded MC159 protein which is a viral FLICE-inhibitory protein (v-FLIP) with two death effector domains and inhibits both Fas- and TNFR-mediated apoptosis , severalHCV also encodes several proteins that antagonize host cell death signals. Although HCV core protein sensitizes Jurkat T cells to Fas-mediated apoptosis, it inhibits Fas-mediated apoptosis via NF-κB activation in particular HepG2 cell lines, suggesting its cell type-specific function . The corThe E3 region of adenoviruses (ADV) encodes several proteins that modulate death receptors on the cell surface and death receptor-mediated apoptosis. The E3-10.4K/14.5K complex selectively mediates loss of Fas surface expression and blocks Fas-induced apoptosis of virus-infected cells , whereasHigh-risk HPV type 16 (HPV16) and 18 (HPV18) play a pivotal role in the pathophysiology of cervical cancer. Like other viruses, HPV has also developed strategies to block host-mediated apoptosis and regulate the survival of infected cells . Some evGlycoproteins of EBOV and Marburg virus (MARV) suppress Fas-mediated apoptosis in Hela cells . HIV-1 NFor many viruses, induction of apoptosis during lytic infection or at late stages of infection may be an important step for the dissemination of progeny virus to neighboring cells while also evading host immune inflammatory and immune responses. With some viruses, inhibition of apoptosis in virus-infected cells can prevent premature death of the host cell and impair virus production, which enables the establishment of viral latency and facilitates persistent infection, contributing to the avoidance of immune surveillance by the host. Therefore, in certain circumstance, either induction or inhibition of death receptor-mediated apoptosis could assist viral infection and contribute to viral pathogenesis.For the host, death receptors can be mediators of the innate immune response to viral infection. The murine and human TRAIL promoters contain interferon regulatory elements and can be activated by interferons, and thus TRAIL is one of the earliest genes induced by interferons ,155. ManIn addition to the role in the cytotoxic activity of innate immune NK cells and pDC cells, death receptor-mediated apoptosis plays an important role in the cytotoxic T cell killing during viral infection. It is well demonstrated that some virus-specific cytotoxic T lymphocytes (CTLs) use the FasL/Fas-dependent lytic mechanism to kill virus-infected or bystander cells, such as lymphocytic choriomeningitis virus (LCMV)-infected cell lysis by LCMV-specific CD4+ CTL , MHC claDeath receptors also mediate apoptosis-independent processes during viral infection. For instance, FasL/Fas system participates in the induction of inflammatory response during virus infection. This has been mainly demonstrated in the context of HSV-2 infection, during which it regulates inflammation in vaginal tissue via the Fas/FasL pathway ,180,181.Death receptor-mediated apoptosis represents a complex and co-evolved mechanism used by the virus and the host, which contributes to viral pathogenesis and host immune surveillance. The infected host cell uses it as part of the antiviral response, whereas the virus appears to balance apoptotic and anti-apoptotic effect to facilitate viral infection. With respect to the potential use of death receptor-mediated apoptosis in the treatment of viral diseases, therapeutic strategies to enhance death receptor-mediated apoptotic clearance of virus-infected cells may be beneficial in some viral infections, whereas in viral infections in which pathogenesis and propagation are enhanced by apoptosis, inhibition of death receptor-mediated apoptosis may be the therapeutic goal. Furthermore, death receptor-mediated apoptosis plays a critical role in the control of virus-infected cells by NK cells, pDCs, and CTLs, which could be the basis for the development of targeted immune control of virus infection. Future studies will need to elucidate in more detail the mechanisms of death receptor-mediated apoptosis by which those immune cells mediate antiviral function. Viral products involved in the induction and suppression of death receptor-mediated apoptosis provide critical insights into cellular apoptotic processes, which could be useful in treating viral diseases. Understanding the mechanism of virally induced death receptor-mediated apoptosis is vital because of its involvement in the pathophysiology of diseases and therapeutic intervention. Given the multifaceted role of death receptor-mediated apoptosis, further preclinical and clinical studies are required in order to determine its specific usage in the treatment of viral diseases.


RNA interference (RNAi), including microRNAs, is an important player in the mediation of differentiation and migration of stem cells via target genes. It is used as a potential strategy for gene therapy for central nervous system (CNS) diseases. Stem cells are considered vectors of RNAi due to their capacity to deliver RNAi to other cells. In this review, we discuss the recent advances in studies of RNAi pathways in controlling neuronal differentiation and migration of stem cells. We also highlight the utilization of a combination of RNAi and stem cells in treatment of CNS diseases. Central nervous system (CNS) diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD), and spinal cord injury (SCI) are characterized by neuronal loss in the brain or spinal cord, which cause corresponding impairments in neural functions ,2,3. TheRNAi is a mechanism to inhibit gene expression by double-strand RNA (dsRNA) . Gene-siStem cells, such as mesenchymal stem cells (MSCs) and neural stem cells (NSCs), are a potentially promising source for cell-based therapy. MSCs can be easily derived from bone marrow , umbilicOther miRNAs, such as miR-29a and miR-9, are also involved in enhancing the neural differentiation of BMSCs. Similar to miR-124, miR-29a also regulates neurogenic markers in BMSCs through targeting REST . It is sThere are many other miRNAs that modulate neuronal differentiation in various types of stem cells. miR-125b, one of the brain-enriched miRNAs, has been identified as a player in regulating neuronal differentiation ,51. In nMiR-506-3p negatively regulates the expression of the transcription factor T-cell factor-3 (TCF3) , which wIt has been demonstrated that different miRNAs could target the same gene and produce synergistic effects. For example, miR-124, -128, and -137 synergistically regulate the overlapping gene Sp1, whose level is decreased as cells differentiate. Silencing Sp1 reduces neuronal differentiation of NSCs and affects their viability and proliferation . Taken tRNAi is considered as an important approach to controlling gene expression . HoweverAlzheimer’s disease (AD) is one of the most devastating, age-related neurodegenerative disorders . The amyNeuronal loss is involved in the pathologies of AD and progressively promotes cognitive and memory impairment . An in vSince neuronal loss is an important feature of AD, one problem is figuring out how to induce neuronal differentiation of stem cells by genetic modification. As stated above, the transcription factor SOX9 is an important regulator in neuronal differentiation of stem cells. Knockdown of SOX9 in stem cells via nanotopography-mediated siRNA induces differentiation of NSCs into neurons more than into glial cells , leadinghtt) gene [Htt plays a crucial role in neuronal fate. It was reported that NSCs with expanded expression of CAG repeats causes neuronal deaths, while silencing the htt gene decreases neurons and increases astrocytes [htt in the animal model of HD improves behavioral and neuropathological abnormalities [htt via RNAi could improve motor coordination and survival in HD mice [htt gene successfully ameliorated the neuropathological abnormalities (insoluble protein accumulation and downregulation of DARPP-32 expression) [htt alleles. It seems necessary to allele-selectively inhibit mutant htt expression. Recently, another study demonstrated that [htt expression in cells derived from HD patients; it also selectively reduced mutant htt allele throughout the brain in a mouse HD model. In addition, allele-selective silencing was induced by targeting the heterozygous single-nucleotide polymorphism (SNP) rs362331 in exon 50 and total htt silencing by miH12 both in vitro and in vivo [htt mRNA lowering in individual neurons, Keeler AM et al. [htt on htt mRNA levels in striatal neurons of Q140/Q140 knock-in mice, another HD model. They found that intrastriatal infusions of AAV9-GFP-miRhtt vectors reduced htt mRNA in striatum through a partial reduction in mutant htt mRNAs per cell in medium spiny neurons. Recently, miRNAs such as miR-10b-5p, miR-128a, and miR-34b-a have been proven to be associated with HD [Htt gene expression is regulated by miRNAs and certain heterogeneous miRNA variants are functional and regulate the same target as canonical miRNAs [htt transferred RNAi to the co-culture U87 cells (previously transduced with mutant htt fragment) and SH-SY5Y cells, leading to decreased levels of mutant htt expressed in the co-culture cells [Huntington disease (HD) is a hereditary, progressive autosomal-dominant neurodegenerative disease caused by an exon 1 CAG trinucleotide-repeat-expansion mutation in the huntingtin (tt) gene ,79. HD itt) gene . The pattt) gene . Htt platrocytes . shRNA-mmalities . Additio HD mice . By examression) . Howeverted that  RNAi by  in vivo . To furtM et al.  investig with HD ,89,90. Hl miRNAs . Taken tl miRNAs . Stem cere cells .Spinal cord injury (SCI) impacts a patient’s physical, psychological, and social well-being due to the traumatic event . ApproxiCurrently, standard therapies only have limited effects on secondary neuronal injury . Thus, sAs mentioned previously, some miRNAs are involved in REST signaling pathways and play a negative role in regulating behavior of stem cells. Therefore, silencing of the REST gene increases expression of mesendoderm differentiation markers  and enhaNogo-66 receptors (NgR) bind to three myelin-associated neurite outgrowth inhibitors , causing inhibition of axon regeneration after injury in the adult mammalian CNS ,108,109.The high proportion of cell apoptosis is one of the main problems in cell transplantation. P75, a neurotrophin receptor, has been proven to induce apoptosis of neurons . P75-supIn addition to neuronal loss, oligodendrocyte cell death and demyelination are also important pathological characteristics of SCI . OligodeBased on the findings summarized above, we propose that stem cells can be used as vectors for transferring RNAi-mediated genetic materials and that RNAi-modified stem cells have potential for treating CNS diseases through various pathways after transplantation . There aThere are multiple pathways affecting the combination of RNAi and stem cells therapies. A better understanding of stem cell biology is needed to help discover different relative genes and develop targeted gene therapies. It has been noted that RNAi can sometimes produce nonspecific, off-target effects, leading to unpredicted phenotypes; this is attributed to miRNA-like binding in the 32 UTRs . For ins


Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105−106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support the development of Fc fusions of GP as a candidate vaccine for human use.Ebola virus (EBOV), a member of the  Filoviridae is a family of zoonotic, filamentous, negative-strand RNA, enveloped viruses consisting of three genera: Ebolavirus and Marburgvirus, which can cause severe hemorrhagic fever in humans and nonhuman primates (NHPs) with high morbidity and mortality rates up to 90% [Cuevavirus, which is pathogenic in bats and was recently discovered in Spain [Marburgvirus is antigenically stable and has a single species with two viruses, Marburg virus (MARV) and Ravn virus (RAVV), whereas Ebolavirus is more diverse and consists of five species, Zaire, Sudan, Taï Forest, Reston, and Bundibugyo ebolavirus each one with a single virus, Ebola virus (EBOV), Sudan virus (SUDV), Tai Forest virus (TAIFV), Reston virus (RSTV), and Bundibugyo virus (BDBV) [The p to 90% –3, and Cin Spain . The rapin Spain , 6. Filos (BDBV) . RESTV is (BDBV) ). It is s (BDBV) .The recent filovirus epidemic caused by a new isolate of EBOV, the Makona strain (EBOV/Mak), started in Guinea in 2013, spread to several countries in West Africa including Liberia and Sierra Leone, and claimed thousands of lives is declared the outbreak officially over in 2015 after a coordinated effort of local and international organizations , 13. TheThe EBOV genome of approximately 19 kb that contains 7 genes: nucleoprotein (NP), VP35, VP40, glycoprotein (GP), VP30, VP24, and the polymerase (L) . TranscrEBOV infection in humans elicits cellular and humoral immune responses  that arWe are currently developing a GP subunit vaccine based on the extracellular domain of GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc). In mammalian cells, EBOVgp-Fc undergoes the complex posttranslational modifications of native GP such as furin cleavage, disulfide bonding of the GP1 and GP2 subunits, glycosylation, and trimer formation . Mice imChinese hamster ovary (CHO) dihydrofolate reductase negative cells  stably transfected with cDNA of the EBOVgp-Fc fusion protein (CHO/EBOVgp-Fc)  were groA plasmid containing the cDNA of the Mayinga EBOV GP (GenBank accession no. AF272001), pVR-1012-ZEBOV-GP , was kinTo rescue replication-competent VSVG-deleted recombinant VSV (rVSV) containing a mucin-deleted EBOV GP, we deleted the mucin-like region of the EBOV GP cDNA between amino acids 312–462 using overlapping PCR . BrieflyWe construct a plasmid to express the extracellular domain of the mucin-deleted EBOV GP fused to the Fc fragment of human IgG1 in cell transfectants. To do so, we used the EBOVgpΔmuc fragment described above and same strategy to construct the plasmid coding for the EBOVgp-Fc fusion protein . The resTo rescue rVSV containing the EBOV GP and GFP genes , we geneTo rescue rVSV contained the mucin-deleted EBOV GP and GFP, we generated a mucin-deleted EBOV GP cDNA followed by the GFP cDNA  as descrReplication competent rVSV in which the VSV-G protein was replaced by the EBOV GPΔmuc, EBOV GP plus GFP, or EBOV GPΔmuc plus GFP were generated using the VSV reverse genetics system , 48 and The FLAG-Fc control protein and the EBOVgp-Fc, a protein containing the extracellular domain of EBOV GP tagged with a DYKDDDDK (FLAG) peptide at the N-terminus and fused to the Fc and hinge fragment of human IgG1 were produced in CHO cell stable transfectants as described . A similth Edition, National Research Council, 2011. The animal facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The USAMRIID Institutional Animal Care and Use Committee (IACUC) approved the animal protocol.Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations following the principles stated in the Guide for the Care and Use of Laboratory Animals, 8Male and female guinea pigs approximately 350–450 g of body weight were used in the vaccination studies. Hartley strain Guinea pigs obtained from Charles River Labs were immunized using vaccine formulated with QS-21  as an adjuvant. Strain 13 guinea pigs obtained from an inbred colony maintained at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) were immunized using vaccine formulated with alum  or poly-ICLC  as adjuvants.th vaccination at day 91. The animals were challenged 14 days after the third boost at day 91 by intraperitoneal inoculation of 1,000 plaque-forming units (pfu) EBOV Mayinga adapted to guinea pigs (EBOV/May-GPA) diluted in PBS [Guinea pigs were vaccinated on day 0, 21, 42, and 63 intramuscularly (i.m.). For the animals immunized using QS21 as an adjuvant, we used 100 μg/dose of EBOVgp-Fc, EBOVgpΔmuc-Fc, or control FLAG-Fc and 25 μg/dose of QS-21. For the animals immunized using alum or poly-ICLC as adjuvants, we used 50 μg/dose of EBOVgp-Fc, or control FLAG-Fc and 500 μg/dose of alum or poly-ICLC. The same number of animals was included in all vaccinated groups (8 guinea pigs/group). Because during the course of vaccination we determined that some animals were pregnant or died due to unrelated causes, these guinea pigs were not included in the challenge studies. Blood samples were collected from the guinea pigs immediately before each vaccination. For the anti-GP pilot study , blood sd in PBS . Guinea d in PBS . Animals5 TCID50 of rVSV-EBOVgp or rVSV-EBOVgpΔmuc produced in Vero E6 cells using serum-free OptiMEM medium. Plates coated with wt VSV were used as the specificity control. After blocked with PBS containing 5% bovine serum albumin (BSA), two-fold dilutions of the guinea pig sera were titrated in duplicates on the virus coated plates. Plates were incubated at 37°C for 1 h, washed and stained with horseradish peroxidase conjugated goat anti-guinea pig IgG  and 3,3',5,5'—tetramethylbenzidine SureBlue TMB substrate . The colorimetric reaction was stopped with 1% sulfuric acid and absorbance was read in an ELISA plate reader at 450 nm. Antibody titers were defined as the highest dilution at which the mean absorbance of the sample was at least two-fold greater than the mean absorbance of the same sera dilution in control wells coated with wt VSV.The levels of EBOV GP-specific immunoglobulin G (IgG) antibodies in vaccinated guinea pigs were determined by a virus particle endpoint dilution ELISA  in 96-we50. It should be pointed out that there is a high degree of correlation between the FRNT50 assay using rVSV-EBOVgp-GFP under BSL-2 conditions and the plaque reduction neutralization test (PRNT) using wild type EBOV/May . To assure comparability between tested groups, we included internal controls using antibodies of known titers, which reacted similarly in assays from the different groups.Anti-EBOV GP neutralizing antibodies were analyzed by a Fluorescence Reduction Neutralization Test (FRNT). To do so, 2,000 pfu of rVSV-EBOVgp-GFP or rVSV-EBOVgpΔmuc-GFP were treated with serum dilutions from the vaccinated animals in the presence of 5% guinea pig serum complement. After incubation at 37°C for 1 h, Vero E6 cell confluent monolayers in 6-well plates were inoculated in duplicates with each neutralization reaction. After adsorption for 1 h at 37°C, the inoculum was removed, cells were washed, DMEM containing 10% FBS was added, and plates were incubated for approximately 12 h at 37°C. Cells were detached by treatment with 1 ml of 0.5 mM EDTA in PBS, washed two times with PBS 2% FBS, and fixed with 2% paraformaldehyde. One million cells from each well were analyzed by flow cytometry for GFP fluorescence using a FACSCanto II cytometer (BD Biosciences). The percent neutralization (% FRNT) of each serum sample was calculated by comparing the number of GFP positive cells in monolayers infected with virus samples treated with pre-immune versus vaccinated serum using the formula 100-(vaccinated/pre-immune) x100. The serum dilution that reduced 50% of the fluorescent cells was defined as FRNT≥0.05, significant (*) when p<0.05, and highly significant (**) when p<0.01.Virus titers were calculated using the ID50 program developed by John L. Spouge . Statistical significance between two groups was determined by unpaired t-test, and also verified by an F-test to compare variances, using the Prism version 6 program . For more than two groups, the data was analyzed by ANOVA to calculate F value with the degrees of freedom and p-value . For multiple adjuvant groups, data was analyzed by ANOVA using the InStat program , and comparison of two treatments was performed using the Bonferroni test. All analysis of IgG responses were carried out on the log-scale. The FRNT50 was obtained using a 4-parameter logistic regression. Kaplan-Meier survival analysis was performed with the Prism version 6 program and evaluated using the Mantel-Cox test. Differences are considered non-significant (NS) when p50 ratio of the different viruses and the amount of GP incorporated into the virus particles, we performed an ELISA using monoclonal antibody KZ52 , we focused vaccine development on EBOVgp-Fc because it contains the mucin region found in the natural forms of GP that may play a protective role in the NHP model and in humans. Hartley strain guinea pigs were vaccinated with 50 μg of EBOVgp-Fc or control FLAG-Fc adjuvanted with alum or poly-ICLC. Total anti-GP antibodies in the sera collected at day 63 just before the last boost were analyzed by the virus particle ELISA . The EBOWe also analyzed anti-GP neutralizing antibodies in the sera from the vaccinated guinea pigs. To do so, rVSV-EBOVgp-GFP was treated with different dilutions of sera and neutralization was evaluated by FRNT50 . The EBOThe guinea pigs vaccinated with alum or poly-ICLC adjuvanted antigens were challenged 2 weeks after the third boost with 1,000 pfu of EBOV/May-GPA. The weight loss in animals vaccinated with EBOVgp-Fc was approximately 10% compared to the approximately 20% in guinea pigs vaccinated with FLAG-Fc vaccine . The weiTo analyze correlates of immunity in our study, we performed ANOVA of the anti-GP antibodies elicited by the EBOVgp-Fc vaccine using the different adjuvants . It shou4.5 to 106 and 102.5 to 103.5, respectively, which is comparable to the antibody titers induced by other vectored and non-vectored vaccine candidates. Therefore, it is likely that the EBOVgp-Fc will also induce high anti-GP titers in NHPs that are commensurate with protection against lethal EBOV challenge as seen with other vectored and non-vectored EBOV candidate vaccines.During vaccine development, EBOV candidate vaccines are sequentially tested in the mouse, guinea pig, and NHP lethal challenge models to determine whether they can progress to clinical trials. Several EBOV GP vaccine candidates are currently under development . EBOV GHere, we analyzed the importance of the adjuvant in conferring complete protection against EBOV/May-GPA lethal challenge in guinea pigs immunized with a protein-based GP subunit vaccine. In this study, we used the extracellular domain of EBOV GP fused to human IgG1 Fc, which may confer stability and enhance immunogenicity of GP, but did not analyze the contribution of the Fc fragment using constructs containing only the extracellular domain of GP. The same Fc fragment was fused to all constructs in this study to allow comparison between adjuvants. We vaccinated animals with the Fc fragment alone formulated with the different adjuvants as control to rule out the possibility of nonspecific protection resulting from immune responses against Fc. In animals immunized with EBOVgp-Fc, the use of poly-ICLC as an adjuvant increased the protective effect of the vaccine conferring complete protection whereas formulation with QS-21 or alum resulted in approximately 65% protection. It should be pointed out that we used 100 μg/dose of EBOVgp-Fc vaccine in the QS-21 group and 50 μg/dose in the alum and poly-ICLC groups. Because the QS21-adjuvanted vaccine elicited lower antibody levels and protection than the alum- or poly-ICLC-adjuvanted vaccines, the dose of EBOVgp-Fc was not responsible for the low performance of the QS21-adjuvanted vaccine. However, the use of strain 13 guinea pigs in the QS-21 experiments compared to the Hartley guinea pigs used in the alum and poly-ICLC experiments may have influenced the level of the antibody response. Statistical analysis revealed that there are no significant differences between the poly-ICLC, QS-21, and alum survival curves, so our data only points to the tendency of poly-ICLC to induce complete protection, which confirmation will require further investigation. The physicochemical characteristics and immune targets of the adjuvants played a significant role in inducing complete protection. Because limitations in space and number of animals per experiment under BSL-4 conditions, we could not evaluate all the adjuvants at the same time, and we did not compare the immunogenicity of GP constructs in the absence of an adjuvant. We first evaluated the effect of QS-21 using the Fc fusion proteins containing the full-length and mucin-deleted extracellular domains of GP. Our data showed that these two constructs induced similar antibody responses and protection levels, so we focused our study on Fc fusion construct containing the full-length extracellular domain of GP and analyzed the effect of the alum and poly-ICLC adjuvants.Quillaja saponaria, is an amphipathic glycoside that acts as a surfactant and binds to cholesterol in biological membranes resulting in pore formation [The adjuvants that we used in this study have very different characteristics. Poly-ICLC, a synthetic polyinosinic:polycytidylic acid (poly-IC) double-stranded RNA stabilized with poly-L-lysine to increase RNase-resistance, is recognized by the cytosolic RNA helicase MDA-5 and the endosomal TLR3 and activate the production of type I IFN that stimulates B, T, and dendritic cells . QS-21, ormation . QS-21 iormation ). Alum  responses . QS-21 iTotal anti-GP IgG antibody levels elicited by VSV and adenovirus vectored EBOV GP vaccines correlated with protection against EBOV/May-GPA lethal challenge in guinea pigs and NHPs . HoweverThe GP structure  suggestsIn humans and NHPs, the human IgG1 Fc fragment present in EBOVgp-Fc could enhance the immunogenicity of the vaccine by interacting with Fcγ receptors on antigen presenting cells –61. The In summary, we demonstrated that the poly-ICLC adjuvanted EBOVgp-Fc subunit vaccine is highly immunogenic and completely protected guinea pigs against lethal EBOV/May-GPA challenge. In mice, we previously showed that EBOVgp-Fc elicited anti-GP total and neutralizing antibodies as well as T-cell immunity that protected animals against lethal challenge with mouse-adapted EBOV. This gu


It is now well established that cancer cells co-exist within a complex environment with stromal cells and depend for their growth and dissemination on tight and plastic interactions with components of the tumor microenvironment (TME). Cancer cells incite the formation of new blood and lymphatic vessels from preexisting vessels to cope with their high nutrient/oxygen demand and favor tumor outgrowth. Research over the past decades has highlighted the crucial role played by tumor-associated blood and lymphatic vasculature in supporting immunoevasion and in subverting T-cell-mediated immunosurveillance, which are the main hallmarks of cancers. The structurally and functionally aberrant tumor vasculature contributes to the protumorigenic and immunosuppressive TME by maintaining a cancer cell’s permissive environment characterized by hypoxia, acidosis, and high interstitial pressure, while simultaneously generating a physical barrier to T cells' infiltration. Recent research moreover has shown that blood endothelial cells forming the tumor vessels can actively suppress the recruitment, adhesion, and activity of T cells. Likewise, during tumorigenesis the lymphatic vasculature undergoes dramatic remodeling that facilitates metastatic spreading of cancer cells and immunosuppression. Beyond carcinogenesis, the erratic tumor vasculature has been recently implicated in mechanisms of therapy resistance, including those limiting the efficacy of clinically approved immunotherapies, such as immune checkpoint blockers and adoptive T-cell transfer. In this review, we discuss emerging evidence highlighting the major role played by tumor-associated blood and lymphatic vasculature in thwarting immunosurveillance mechanisms and antitumor immunity. Moreover, we also discuss novel therapeutic approaches targeting the tumor vasculature and their potential to help overcoming immunotherapy resistance. Cancer cell and stromal cell interface enforces a tumor microenvironment (TME) that is permissive for tumor growth.The dynamic properties of the TME regulate how malignant cells respond to therapy.Cancer cell-derived proangiogenic factors triggers unproductive angiogenesis and lymphangiogenesis that facilitate tumor growth and metastasis.The structurally and functionally abnormal tumor blood and lymphatic vasculature favor escape of malignant cells from antitumor immunity and fosters the immunosuppressive TME.Endothelial cells (ECs) of the tumor vasculature actively suppress antitumor immunity by regulating recruitment, adhesion, and function of immune cells and by inducing killing of effector T cells.A complex bidirectional interface between tumor vasculature and the immune cells regulates therapy responses.Targeting the tumor vasculature with antiangiogenic agents allows a transient improvement of the vessels that improves tumor oxygenation and enhances drug delivery, immune cells' infiltration, and immunotherapy efficacy.What are the molecular mechanisms regulating the intense crosstalk between ECs and immune cells within the TME?What is the role of other stromal cells  in tumor angiogenesis?Which vasculature-targeting approaches can ‘heat up’ the TME and favor infiltration of T cells?Which tumor vasculature-targeting regimens create the best window of opportunity required for a durable effect on immunostimulating TME?Which pathway and EC-specific molecular target should we target to improve therapy responses?How should the lymphatic system be targeted considering that it serves peripheral tolerance but also facilitates adaptive immune response by draining tumor-associated antigen(-presenting DC)?What are the best treatment scheduling options for antiangiogenic therapies when combined with immunotherapy modalities?Do tumor vessel-normalizing strategies offer a best treatment strategy to improve T-cell function and immunotherapy?Does the concept of vessel normalization extend to the lymphatic vasculature and what are the underlying mechanisms?Do vessel-normalizing strategy in combination with immunogenic cell death-based approaches synergize?Which biomarkers will allow monitoring the effects of vessel normalizing drugs on patient’s immunological responses to therapy?1 and include fibroblasts, cells of the immune systems, pericytes, and ECs of the blood and lymphatic vasculature.In recent years, tumors have been recognized as complex dysorganized and chaotic organs, where cancer cells co-exist and co-evolve with their stroma. This view is a major shift from the previously accepted ‘cancer cell-centered’ perception of cancer evolution, which mainly focused on understanding oncogenic drivers and cell-autonomous features of cancer. It is now increasingly accepted that the interface between malignant and non-transformed cells defining the tumor microenvironment (TME), represents a highly plastic tumor ecosystem that supports tumor growth and dissemination through the various stages of carcinogenesis. Apart from cancer cells, the TME of a solid tumor contains a complex interstitial extracellular matrix and various stromal cells that are recruited from the surrounding tissues or from the bone marrowWithin the TME, cancer cells thrive and maintain a dynamic communication with all TME components through the release of soluble factors  or through cancer cell–stromal cell contacts, which ultimately drive a chronic inflammatory, immunosuppressive, and pro-angiogenic niche that promotes dissemination of cancer cells and thwarts the effects of various therapeutic interventions, including immunotherapy. Moreover, this intersection is bi-directional, since each stromal component of the TME may establish a proficient interface with cancer cells, which facilitates cancer progression, at virtually any stage of tumorigenesis.3. Distinguishing mechanisms enabling an immunoevasive cancer cell phenotype include a reduced immunogenicity due to loss in expression of tumor-associated antigens (TAAs) or major histocompatibility complex (MHC) class I molecules, acquired DNA copy number alterations and oncogenic signaling, upregulation of cellular immune checkpoints like programmed death ligand 1 (PD-L1), indoleamine 2,3-dioxygenase (IDO), and tryptophan 2,3-dioxygenase (TDO), and altered metabolism resulting in a low pH and secretion of various metabolites4. Moreover, through increased production of immunosuppressive and tumor-promoting cytokines, cancer cells modulate the polarization, activity, and expansion of various immune cell subpopulations and interface with ECs, causing alterations in their structural integrity and functional properties, thus diminishing antitumor immune responses.For example, a large body of experimental evidence supports the concept that the immune system is able to eradicate emerging tumors through the process of cancer immunosurveillance before cancer cells evolve the ability to erode detection and eradication by immune cells+ T cells can recognize TAA presented through a MHC class I molecule on cancer cells and induce their killing via the perforin-granzyme and/or Fas ligand (FasL)/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) systems, although this depends on the degree of functional inhibition by the TME and the presence of immunosuppressive regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). In this scenario, the blood and lymphatic vasculature have important roles as physical and functional barriers for tumor-infiltrating immune cells and TAA/TAA-presenting DC drainage to the LNs, respectively.In fact, cancer immunosurveillance, which is driven largely by activated effector T cells, is impaired at different levels by several obstacles imposed by the increasingly hostile TME. To exert their function, T cells need to be properly activated by antigen-presenting dendritic cells (DCs), usually by encountering DCs in peripheral lymph nodes (LNs), egress the LNs and home to the tumor and finally extravasate from blood vessels and infiltrate the tumors. Thereafter, activated CD8Finally, such an intense crosstalk between cancer cell and stromal cells not only promotes tumor growth and dissemination but also gravely affects the efficacy of multimodal anticancer treatment. This is particularly true for the currently, clinically used cancer immunotherapies, such as those employing immune checkpoint inhibitors (ICIs) or adoptive T-cell transfer (ACT), that primarily aim to reinvigorate antitumor T-cell activity.Here we aim to discuss the current view on the cancer cell-induced alterations in the blood and lymphatic vasculature as well as (sentinel) LNs that profoundly impede antitumor immunity. We also summarize the advances and therapeutic combinations targeting the tumor vasculature that may overcome immunotherapy resistance.5. The formation of a novel vascular sprout is a dynamic and tightly orchestrated process that involves the coordinated action of the highly invasive and motile tip cells at the leading edge -A–vascular endothelial growth factor receptor-2 (VEGFR-2) axis) and the underlying proliferating stalk cells, elongating the sprout and generating the lumen. Such fully formed vessel recruits pericytes and vascular smooth muscle cells thereby promoting stability, integrity, and blood perfusion  required for their high energy demand and growth. Tumor angiogenesis entails the development of new blood vessels from established vascular beds and as such is different from vasculogenesis  or vasculogenic mimicry (the ability of tumor (stem) cells to form vessel-like networks)8. In fact, not only cancer cells secrete high amount of VEGF and can contribute to VEGF-independent angiogenesis , VEGF-C, VEGF-D, and platelet-derived growth factor (PDGF)-C) but they can also respond in an autocrine or paracrine manner to prosurvival and prometastatic VEGF signaling5. Although tumor angiogenesis is meant to support blood supply to the tumor, the resulting vessel network is leaky, chaotically organized, immature, thin-walled, and ill-perfused  and interleukin (IL)-8. These cytokines become ubiquitously abundant in the TME and overwhelm angiostatic signals, such as angiostatin and endostatin, thereby inducing a pro-angiogenic switchsed Fig. . This un9, is associated with structurally immature vessels that are unstable, leaky, and tortuous. Ill-covered perfusion results in diffusion-limited nutrient delivery . Moreover, due to the high interstitial fluid pressure (IFP) in the tumor (a result of vessel leakiness) these vessels are prone to collapse and diminish the perfused tumor area  and acidic (due to increased anaerobic glycolysis of cancer cells) TME that facilitates the selection of cancer cells with genetic  and epigenetic alterations that enhance their aggressiveness. Importantly, hypoxia and acidosis  levels. These factors collectively induce FasL expression on tumor ECs, which upon binding to Fas expressed on T cells, triggers their killing by apoptosis. Due to a differential expression of c-FLIP , CD8+ T cells are more adversely affected by these events than Tregs12. Moreover, tumor-associated ECs can preferentially promote the recruitment of Tregs by the upregulation of the multifunctional endothelial receptor CLEVER-1/stabilin-1, thus suggesting that tumor endothelium can support both the recruitment and the survival of immunosuppressive T cells13. In addition, VEGF-A mediates a clustering defect of the adhesion molecules like intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion protein (VCAM)-1, which hampers immune cell extravasation14. Thus, aside from stimulating angiogenesis, VEGF-A also contributes to the impediment of an efficient EC–lymphocyte interaction. Furthermore, the endothelin B receptor -1-regulated endothelin-1)) on tumor ECs is implicated in counteracting T-cell adhesion as neutralization of ET-BR increases tumor-infiltrated lymphocytes (TILs) and improves responsiveness to immunotherapy15. Although these adhesion molecules can bind multiple leukocyte subtypes, it is still unclear which compensatory signals maintain the intratumoral presence of monocytes and neutrophils.A hypoxic TME is associated with high VEGF-A, IL-10, and prostaglandin E16, and it interferes with TNF-induced nuclear factor-kB activation 17.Hypoxia-induced signaling mediates the presence of certain immunosuppressive immune cell types, including immature DCs, TAMs, and tumor-associated neutrophils (TANs), as well as, MDSCs. VEGF-A is associated with reduced DC differentiation from hematopoietic progenitors2) stimulate the M2 phenotype18 and the expansion of monocytic (CD11b+) and granulocytic (Gr1+) MDSCs19. MDSCs are a source of transforming growth factor (TGF)-β, a crucial immunosuppressive factor, in the TME20. Importantly, these TAMs and Gr1+ myeloid cells can also render tumors non-responsive to VEGF/VEGFR inhibition (as mentioned later) and induce angiogenic relapse21.HIF-1 targets, VEGF-A and IL-8, aid the recruitment of immature myeloid cells that may stay undifferentiated (and develop into MDSCs) or develop into TAMs. TAMs have a high level of plasticity displaying either pro-inflammatory features (M1 phenotype) or immunosuppressive features (M2 phenotype). Hypoxia-associated molecules -mediated responses23. Moreover, TGF-β induces IL-receptor-associated kinase (IRAK)-M expression in TAMs, important for an M2 phenotype, that has relevant implications as the growth rate of transplanted Lewis Lung carcinoma (LLC) cells was reduced in IRAK-M−/− mice24. Regarding TANs, TGF-β can induce the protumorigenic N2 phenotype25, although it is not clear to what extent N2 cells exert long-term protumor effects since neutrophils have a relatively short life span after they leave the bone marrow and are particularly sensitive to nutrient deprivation, as typically found in tumors26.In addition, the generated mechanical stress (due to high IFP) leads to TGF-β production from fibroblasts  ligand-28 (CCL-28) by cancer cells recruits Treg29. The MDSCs that are attracted and expanded during hypoxic conditions can produce limited amounts of reactive nitrogen species  that can cause nitration of tyrosines in a T-cell receptor (TCR)–CD8 complex (that impedes interaction with antigen–MHC complexes30) and some chemokines like CCL-2. Importantly, nitrated CCL-2 can still serve as a chemoattractant for monocytes (that can function as MDSCs within a tumor) but not effector T cells31.Immature DCs may express immunosuppressive molecules, such as IDO and TDO. IDO converts the essential amino acid tryptophan in the extracellular matrix to kynurenine. Low tryptophan levels starve effector T cells while favoring T32 that inhibits the effector function of T cells (thereby inducing T-cell anergy). PD-L1/PD-L2 are commonly expressed by cancer cells, tumor-associated ECs, macrophages, fibroblasts, and DCs33. Moreover, VEGF-A-enhanced expression of PD-1, T-cell immunoglobulin mucin (TIM)-3, and cytotoxic T lymphocyte antigen-4 (CTLA-4) on intratumoral CD8+ T cells34 increases susceptibility to functional inhibition, thereby invigorating T-cell exhaustion. Furthermore, the acidic nature of the TME also inhibits the induction of functional CTLs from memory T cells35.Additionally, immune checkpoints can modulate the functional activity of different T-cell populations. In this regard, the HIF-1 pathway is a major inducer of PD-L1/PD-L2 expressionCollectively, these data highlight that the tumor vasculature is a crucial TME compartment with the ability to suppress both directly  and indirectly  antitumor immune responses.Besides blood vessels, the vascular network includes the lymphatic system. The lymphatic vessels (LVs) drain interstitial fluid consisting of a plethora of proteins, lipids, and cells from a tissue  and actively contributes to cancer cell dissemination39. Moreover, LECs function as antigen-presenting cells and induce immunological tolerance and promote the apoptosis of tumor-reactive CTLs40.In the TME, cancer cell-derived ligands of VEGF receptor (VEGFR-)3  can induce lymphangiogenesis, the equivalent of blood vessel angiogenesis that leads to the sprouting and attraction of LVsHere we discuss some of the most salient features linking tumor-associated lymphatics to the regulation of antitumor immune responses in the TME, using melanoma as a paradigm of immunosuppressive and aggressive cancer harnessing the lymphatic system for dissemination Fig. .Fig. 3Th+) infiltration, including immunosuppressive subtypes  and antitumor subtypes . Consistently, when testing two independent mice models (including a transgenic K14-VEGFR-3-Ig model) that reduced peritumoral LYVE1-positive dermal lymphatic capillaries in a B16-F10 melanoma, general immune cell tumor infiltration declined including the number of Treg, inflammatory monocytes and CD8+ T lymphocytes. Moreover, this phenotype was associated with decreased DC trafficking from tumor to dLN. This indicates that, whereas lymphatic capillaries are required for T-cell infiltration to occur, they can also cause unproductive adaptive antitumor response41. Moreover, LECs can present self-antigens on MHC-I proteins to promote tolerance that is accentuated by secretion of immunosuppressive chemokines ), high PD-L1/L2 expression, and suboptimal co-stimulatory protein levels (CD80/CD86). Although LECs express basal levels of PD-L1 thereby modulating peripheral tolerance, PD-L1 expression is increased in tumor-associated LECs42, likely through HIF-1 or interferon (IFN)-γ, which are potent inducers of PD-L1/L2 expression in LECs. Thus, in case of successful tumoral infiltration by active CTLs, tumor-associated LECs may attenuate effector T cells’ cytolytic activity. Importantly, LECs increased PD-L1 expression when pulsed with a peptide of the model antigen ovalbumin (OVA). In the presence of PD-L1 blockade, co-culturing these LECs with OT-1 CD8+ cells resulted in improved cancer cell killing by OT-1 cells42, thus disclosing a LEC-mediated mechanism through which ICIs might stimulate CTL activity.A lymphatic score -1, and VEGF-C) in metastatic cutaneous melanoma patient samples correlated positively with immune cell (CD4543) and causes an increase in lymphatic pump activity 44. Thus VEGF-C can increase the tissue drainage of cells and TAAs. Additionally, MDSCs at the sites of inflammation are potent NO producers that impair lymphatic flow45; however, it is not clear to what extent this contributes in tumors. Still, cancer cells  secrete cytokines and chemokines that recruit myeloid cells  and could thus impair these contractile cycles and the drainage of TAAs/TAA-presenting DCs to dLNs. Moreover, reduced lymph drainage and the lack of  intratumoral LVs contributes to the high IFP43 and subsequent immunosuppressive effects. Improving the lymphatic vessel function as well as reducing the intratumoral MDSCs are seemingly important targets in improving antitumor immunity.The lymphatic capillaries drain to larger contractile vessels referred to as collecting lymphatics that guide lymph toward dLNs. These regulate the lymphatic flow by contractions of surrounding smooth muscle cells. This is established by a spatiotemporally regulated NO production by LECs. Tumor-derived VEGF-C attracts LVs into the tumor , IL-2 (B and T-cell proliferation stimulus), and granulocyte macrophage colony-stimulating factor (DC maturation factor)47. Thus, initially, an immune response is incited in a dLN that can also be sufficient to prevent colonization. Yet the tumor eventually overcomes this protective effect. In agreement with this, in the presence of a subcutaneous B16-F10 tumor, intralymphatic B16-F10 injection resulted in effective tumor growth46. This can be a result of tumor-derived secreted factors and immature DCs and recruitment of MDSCs. Regarding the former study using an OVA-expressing B16-F10 melanoma model, additional VEGF-C overexpression led to reduced IFN-γ-producing CD8a+ OT-1 cells in the dLN, possibly due to enhanced lymph flow and LEC-mediated tolerogenic events48. Moreover, in a different melanoma model (B16-F1), CCL-21 expression in dLNs reduced progressively in time after tumor injection, as compared to unchallenged LNs49. This could possibly result in an impaired T-cell retention, which enables clonal expansion before LN egress50.In essence, the LN is a tissue for the recognition and presentation of antigens to prime or tolerogenize adaptive immune responses. A tumor drains various secreted factors that influence the LN microenvironment in favor of immunosuppression. This counteracts antitumor immunity and generates a hospitable environment for the seeding and growth of cancer cells (“lymphovascular premetastatic niche”). In line with this, B16-F10 cells injected into the LN, but not subcutaneously, are rejected in a CD8Thus the lymphatic system can support (draining of TAAs/TAA-presenting DCs) as well as attenuate (tolerogenic events) antitumor immune responses.28. Briefly, immunotherapy aims to modulate the host’s immune system to promote antitumor immunity and it broadly includes treatments with cytokines/immunomodulatory drugs, monoclonal antibodies (mAbs), adoptive cell transfer, and anticancer vaccines, such as DC vaccines53. However, the current landscape of cancer immunotherapy is largely dictated by ICIs, principally because of their clinical success and prominent and durable responses in patients of several histological tumor types56. The most prominent ICIs are mAbs blocking the activity of CTLA-4, PD-1, and PD-L1. Emerging evidence, moreover, highlights that the type of cancer cell death may favor or impede antitumor immunity and regulate the success of ICIs in combinatorial regimens57. Indeed, antitumor immunity can be accentuated via the induction of immunogenic cell death (ICD) in cancer cells, thus acting as ‘in situ’ vaccines59. Major hallmarks of ICD are the ER stress-regulated and spatiotemporally defined emission of danger signals 61. Moreover, ICD is uniquely associated with ‘altered-self mimicry’ elicited by type I IFN cytokines (consisting of IFN-α and IFN-β) and a pathogen response-like chemokine signature (consisting of C-X-C ligand (CXCL)-1, CCL-2, CXCL-10, or homologs thereof)63. Of note, cancer cells succumbing to ICD can also be used for creating next-generation DC-based vaccines64.The discussion so far establishes that the tumor vasculature (both ECs and LECs) is an essential regulator of the intersection between cancer cells and immune compartment within the TME. By extension, tumor vasculature can henceforth play an important role in regulating responses to cancer immunotherapy66. These are in large part caused by the profoundly immunosuppressive TME and cancer cell-autonomous mechanisms of immunoevasion  . Compelling evidence indicate that spatial, functional orientation and density of T lymphocytes within the tumor is associated with good patient prognosis across many cancer types69.Although immunotherapy has prolonged survival of many cancer patients , there are still various hurdles limiting its therapeutic efficacylls Fig.  over immBased on these emerging lines of evidences, we surmise that targeting of tumor vasculature might improve the efficacy of cancer immunotherapy. In fact, this is one of the main reasons behind recent proposals to target the tumor vasculature in combination with cancer immunotherapy. In the next section, we describe and discuss some potential combinatorial strategies using antiangiogenic and immunotherapy approaches.70. Later on, preclinical studies showed that vessel pruning leads to an increase hypoxic (but not ischemic) tumor areas71, which in turn supported tumor growth and metastatic dissemination. Indeed, hypoxia induced by anti-VEGF/VEGFR therapy may be in part responsible for the angiogenic relapse and therapy resistance observed after vessel blocking strategies, which may involve distinct immunosuppressive immune-cell populations, including Gr1+CD11b+ and TAMs72. A recent study showed that these myeloid cells are recruited by the cancer cell-derived, angiostatic chemokine, CXCL14, which instigated PI3K signaling in these myeloid cells. In line, inhibition of this pathway was required for the durable effects of antiangiogenic therapy21.Targeting of the VEGF/VEGFR axis has been the most preferred combinatorial approach for immunotherapy-related studies. Initially, monotherapy with antiangiogenic agents, such as the anti-VEGF antibody bevacizumab, by blocking the VEGF/VEGFR-dependent survival and growth of the blood vasculature, was thought to starve the tumor thus halting tumor progression and improving patient survival. In spite of promising initial preclinical results, this vessel-targeting therapy, called vessel blocking, did not elicit the expected results in cancer patients and failed to show substantial improvements in response rates or survival benefits73. Another study showed that DC-101  treatment of glioma xenografts increased vessel normalization that is associated with a time window for the synergistic effect of the combined treatment with radiotherapy, an inducer of ICD71.However, in experimental mouse models, Bevacizumab  treatment resulted in a transient remodeling of the tumor vasculature by increasing the number of matured  vessels, decrease permeability, reduce IFP, and increase perfusion in neuroblastoma xenografts. This vessel ‘normalizing’ effect was transient as the observed intratumoral penetration of topotecan and etoposide only improved the first days after Bevacuzimab treatment74.Hence, these data suggest a window of opportunity to establish a synergistic effect between tumor vessel-normalizing agents and immunotherapy76. Moreover, the resulting improvement in tumor oxygenation may increase the efficacy of immunotherapy. Indeed, as mentioned above, hypoxia and poor intratumoral infiltration of T cells, caused by the poor perfusion of the aberrant tumor vessels, attenuate the efficacy of ICI-based immunotherapy77. Opposite to this, hyperoxygenation increases CTL activity and correlates with improved clinical responses to ICIs78.They also suggest that ‘normalizing’ or ‘healing’, rather than destroying, the erratic tumor vasculature may restore normal structural functional aspects of the vessels, which elicit a better therapeutic outcome. In line with this, ‘vessel normalization’ by improving vessel functionality results in better perfusion of the tumors and by increasing the transporting capability of vessels improves both drug delivery  and therapy responses, which strongly depend on adequate tumor blood supply79. In addition, DC-101 treatment led to tumor vessel normalization and reduced tumor hypoxia only in low (10–20 mg/kg) but not high dose (40 mg/kg) treatments. Moreover, this was accompanied with important changes in the TME with a shift toward tumor-suppressing Th1-mediated immune responses, including TAM polarization to an M1-like phenotype and increased CD4+ and CD8+ T-cell tumor infiltration. These changes in the TME also enhanced the effect of vaccine-based immunotherapy80. In addition, transient targeting of VEGF/VEGFR axis may reverse DC maturation defects81 and lower VEGF-A induced PD-1, TIM3, and CTLA-4 expression on CD8+ T cells34; however, Bevacizumab may also inhibit the phagocytic ability of DCs and macrophages82.In the context of immunotherapy, DC-101 treatment also associated with an increased B16-F10 melanoma infiltration of adoptively transferred T cells and an enhanced tumor growth delay84. Recently, the antimalarial compound and first-generation autophagy inhibitor chloroquine (CQ) was found to elicit in vivo vessel normalization through the activation of the Notch-signaling pathway in blood ECs85, leading to a more quiescent EC phenotype86. Both the tumor vasculature-normalizing and antimetastatic effects of CQ were completely blunted when melanoma cells were implanted in mice lacking Notch1 in ECs. By normalizing the abnormal tumor vasculature, CQ attenuated tumor hypoxia and caused the generation of a more EC solid barrier that impeded cancer cells’ intravasation and metastasis86. Intriguingly, a recent preclinical study showed that, in spite of its mild immunosuppressant effects, CQ does not impair antitumor immunity in vivo and can synergize with immunotherapy88. Another therapeutic strategy may entail reprogramming the ECs' glycolytic phenotype. Recent studies revealed that ECs depend predominantly on glycolysis for ATP production. Furthermore, this glycolytic phenotype is aggravated in the TME by the enhanced VEGF signaling and contributes to vascular dysfunction90.Besides blocking the VEGF/VEGFR axis, other strategies have been shown to induce vessel normalization91. Thus pharmacological inhibitors targeting EC glycolytic metabolism could reverse tumor-induced alterations in ECs leading to a vessel normalization phenotype92, a therapeutic strategy warranting further experimental and clinical confirmation validation.A recent study showed that blockade of the key glycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 normalized blood vessels, an effect that was associated with a tightened vascular barrier (fewer metastases) and increased perfusion (improved chemotherapy efficacy)95 or, oppositely, certain miRNAs affect the VEGF/VEGFR pathway )96 to modulate angiogenesis. Interestingly, cancer cell-secreted vesicles containing miR-494 can promote angiogenesis in ECs97. Thus, as tumor-associated conditions can promote the expression of miRNAs to support the highly angiogenic TME (either cell autonomously or via cross-communication), miRNAs could be considered as potential targets of antiangiogenic/vessel-normalizing approaches. Nevertheless, this is still an emerging field that requires further research to reach a better understanding of how (specific) targeting miRNAs may enhance immunotherapy efficacy.Moreover, recent insights show the relevance of non-protein-coding micro-RNAs (miRNAs) in angiogenesis (see refs. 98) besides establishing immunosuppressive events (tryptophan depletion). Interestingly, in the context of immunotherapy, IDO inhibition synergizes with ICI approaches in preclinical models99, which may therefore be contributed through IDO-mediated effects on tumor vasculature. Furthermore, secretion of galectin-3 (whose expression is induced by hypoxia and nutrient deprivation) inhibits the effector function of CD8+ T cells100 and also invigorates VEGF and bFGF-induced angiogenic events in ECs101. Therefore, targeting these crosstalks and signaling axis could shape a TME in favor of antitumor immunity; however, these possibilities need further investigations and validation in preclinical models.Aside from VEGF-A, other proteins that promote immunosuppression and angiogenesis may be interesting targets. IDO can stimulate angiogenic events , whereas an ACT approach (OT-1 cells activated with OVA-peptide-loaded DCs) was more effective (possibly due to reduced immunosuppressive TME)41.Despite only few studies focusing on targeting the tumor-associated lymphatic structures, its relevance for immunotherapy outcome should not be underestimated. A study utilizing a B16-F10-OVA model showed that VEGF-C overexpression was able to protect against the antitumor immune response elicited by OVA vaccinationTaken together, the aforementioned studies suggest that targeting angiogenesis, with vessel-normalizing strategies in particular, can improve the efficacy of immunotherapies.103. New approaches have been developed to restrict the dose required for a beneficial therapeutic effect on the tumor. Treatment of colorectal cancer-bearing mice with TNF-α or IFN-γ conjugated to the tumor vascular homing peptide TCP-1 resulted in tumor growth delay, increased TUNEL  staining in the tumor, and reduced systemic toxicity compared to unconjugated cytokines. Importantly, the TME also improved as the CD8+ (TCP-1/TNF-α) and CD4+  cell infiltration increased104 and the vasculature normalized (TCP-1/TNF-α)105. TCP-1/TNF-α improved 5-FU delivery and, due to the synergistic effects, improved drug-induced tumor control105. In addition, conjugating TNF-α to a Cys-Asn-Gly-Arg-Cys (NGR) peptide (recognizing an aminopeptidase N (CD13) isoform on tumor-associated ECs) led to increased adhesion molecule expression on ECs, increased CD8+ T cell infiltration in B16-OVA melanoma, and improved outcome of both ACT (with OVA-specific in vitro-activated OT-1 cells) and DC-OVA vaccine approaches106. Other approaches use a small immune protein (L19) to target the additional extra-domain (ED-B) of fibronectin associated with tumor neovasculature. Combined with either dacarbazine107 or radiotherapy109, L19-IL2 treatment enhanced the efficacy of the therapy modality, which was suggested to be CD8+ T-cell dependent108 possibly due to the ICD-inducing ability of radiotherapy that enhances the CD8+-mediated immune response.Administration of antitumor immunity-stimulating cytokines such as IL-2, TNF-α, and IFN-γ can be beneficial; however, it is limited by maximum tolerated doses in patients110) is one of the main predictor of therapy responses in patients, this has generated the therapeutic perspective of targeting the abnormal tumor vasculature to relieve critical TME-associated conditions that antagonize the efficacy of immunotherapy. In line with this, an increasing amount of preclinical data indicate that vessel-normalization strategies, eliciting a transient improvement of the aberrant structural and functional features of the tumor blood vessels, results in lowering tumor hypoxia and increasing drug delivery, thereby enabling immune cell infiltration and synergize with immunotherapies for more durable effects111. These findings have important implications for the design of a combinatorial strategy using vessel-normalizing agents with immunotherapy. However, there are many outstanding questions and challenges that remain to be addressed.To maintain a cancer cell permissive and immunosuppressive microenvironment enabling tumor growth and dissemination, cancer cells educate and corrupt stromal cells. Emerging evidence indicate that cancer cell-induced effects on both the blood and lymph endothelium are crucially involved in the generation and maintenance of an immunosuppressive TME. In particular, the tumor vasculature can actively suppress antitumor immune responses by providing a barrier to T cells' infiltration in the tumor, by selectively killing T cells, or by increasing tolerogenicity against TAAs. Given that spatial, functional orientation and density of T lymphocytes within the tumor  should favor their future applications. Also, the role of other stromal cells, like cancer-associated fibroblast should be considered as these can promote angiogenesis as well. Given the emerging relevance of the dynamic intersection between the immune cells  and ECs, in angiogenesis, and relapse after antiangiogenic therapy, more studies are needed to reveal potential targets that blunt the recruitment of immunosuppressive immune cells fostering tumor regrowth. Further, when applying tumor vasculature targeting regimens, the effects of additional modulation of the lymphatics  should be carefully considered, since whether modulation of lymphangiogenesis overcomes tolerogenic events or impairs stimulation of an adaptive response remains still ill-defined. Moreover, whether the concept of vessel normalization can be extended to lymphatic vessels is still elusive.In conclusion, targeting the tumor vasculature to induce vessel normalization may provide a promising strategy to optimize the efficacy of currently employed immunotherapies as it could lower the level of immunosuppression in the TME. Yet, it is clear that, if we want to exploit the full potential of the immune system to cure cancer, we will have to act at multiple levels in order to ‘normalize’ the TME.


The symmetric tissue and body plans of animals are paradoxically constructed with asymmetric cells. To understand how the yin-yang duality of symmetry and asymmetry are reconciled, we asked whether apical polarity proteins orchestrate the development of the mirror-symmetric zebrafish neural tube by hierarchically modulating apical cell-cell adhesions. We found that apical polarity proteins localize by a pioneer-intermediate-terminal order. Pioneer proteins establish the mirror symmetry of the neural rod by initiating two distinct types of apical adhesions: the parallel apical adhesions (PAAs) cohere cells of parallel orientation and the novel opposing apical adhesions (OAAs) cohere cells of opposing orientation. Subsequently, the intermediate proteins selectively augment the PAAs when the OAAs dissolve by endocytosis. Finally, terminal proteins are required to inflate the neural tube by generating osmotic pressure. Our findings suggest a general mechanism to construct mirror-symmetric tissues: tissue symmetry can be established by organizing asymmetric cells opposingly via adhesions.  •Apical polarity proteins localize in a pioneer-intermediate-terminal order•The orderly localized proteins orchestrate apical adhesion dynamics step by step•Apical adhesions assemble asymmetric cells opposingly into a symmetric tissue Ichthyology; Developmental Neuroscience; Evolutionary Developmental Biology HoweverVertebrate neurulation occurs via either the “epithelium-wrapping” mode, in which an epithelial sheet wraps around pre-existing apical extracellular space as an interior lumen, or the “lumen-inflation” mode, in which epithelial cells aggregate to first form a solid rod from which an interior lumen subsequently emerges . Proper N-cadherin (N-Cad) mutants display defects starting at the neural keel stages (Nagie okom520 (nokm520) mutants, in which the homolog of mammalian pals1 and Drosophila stardust genes , Crb2a, Nok, aPKC, Pard3, and Nan: N-Cad , Claudinn: N-Cad , Pard3 , then enrich apically at the early neural rod stages (around 14-ss), and afterward persist at the apical regions . However+/K+-ATPase α enriched at the apical regions, with a much weaker presence on the lateral membranes  after the pioneer proteins had already localized apically and after neuroepithelial cell proliferation had switched from cross-midline cell division (C-division) mode to parallel-midline cell division (P-division) mode . The finThe progress of the three-step localization of apical polarity proteins is correlated with the changing midline alignments of the apical cell surfaces, as highlighted with apical marker ZO-1 and F-actin bundles. First, when the pioneer proteins become enriched apically at the early stages of the neural rod (∼14-ss), the apical surfaces align jaggedly around the midline C and 1G;We, thus, next examined under transmission electron microscopy (TEM) the apical cell-cell adhesions at different developmental stages. At the neural keel stages, before pioneer proteins enrich apically (∼5-ss), no typical electron-dense cell-cell adhesion complexes appeared at the apical ends of the cells; furthermore, the apical ends of over 50% of cells (42 cells in two embryos) protruded into the opposing half of the tissue, far beyond the midline C, and noSubsequently, at the smooth late neural rod stages (around 18-ss), after intermediate proteins localize to the apical surface, the apical surfaces were smoothly aligned to flank the midline. At this stage, the PAAs remained prominent and the left and right PAAs aligned in two closely positioned but segregated vertical planes; by contrast, the OAAs became diminishing, and the opposing apical cell membranes became segregated by small fragmented lumens . By contrast, the OAAs transiently join the cells between the left and right halves at the midline in opposing orientations at the early neural rod stages.In summary, the PAAs The PAAs and OAAs emerge when pioneer proteins enrich apically and the OAAs dissolve when the intermediate proteins localize apically G. These en face distributions of pioneer proteins N-Cad, ZO-1, β-catenin, and actin bundles at the apical regions of neuroepithelial cells at 14-ss with GFP-labeling-assisted serial sagittal immunomicroscopy  and Nok (N = 105), although not all intermediate protein foci displayed significant Na+/K+-ATPase . We found that these proteins were closely associated with the apical edge of ZO-1 foci E. For ex+-ATPase F.+/K+-ATPase also mediates cell-cell adhesion with their β subunit's homophilic trans-adhesion capability, which is referred to as Na+/K+-ATPase-based adhesion  Thadhesion F; we thuAt the tissue architectural level, the coincidence between the apical enrichment of the pioneer proteins and the formation of mirror-symmetric early neural rod made us wonder whether the pioneer proteins initiate the mirror symmetry, which is subsequently maintained by the intermediate proteins. To test this hypothesis, we next assessed the effects of loss-of-function mutations of N-Cad, Nok, and Pard6γb on mirror symmetry development at 26-ss and 34 hpf (hours post fertilization) (∼12 hr after 26-ss) by examining the distribution of apical marker ZO-1 and basal marker GFAP as well as lumen formation.N-Cadm117 mutants, no apparent midline ventricles developed at the tissue level at the neural keel, rod, and tube stages, and the neural tissue was 1.5–2 times wider than in the wild-type nokm520 and pard6γbfh266 single mutants and nokm520/pard6γbfh266 double mutants , suggesting that cells could not converge effectively toward the midline pt137 and Tg(HSP70:N-Cadm117-GFP)pt136 to examine the effects of expressing N-Cadwt-GFP and N-Cadm117-GFP at 1, 5, 10, or 14-ss on the symmetry formation in N-Cadm117 mutants. Results showed that inducing N-Cadwt-GFP but not N-Cadm117-GFP at 1-ss best rescued the mirror symmetry pt137, localized ubiquitously on the cell membranes at 5-ss and gradually enriched at both the PAAs (ZO-1 positive) and the opposing apical surfaces by 14-ss; then at 18-ss, N-Cadwt-GFP diminished at the opposing apical surfaces but remained enriched at the PAAs and was weakly present on the lateral membranes ; however, at 18-ss, the left and right PAAs segregated from each other into two midline-flanking planes, giving the appearance of smooth alignment, which suggests that left and right halves have separated from each other, and the surface tension makes the apical surface smooth and the PAAs flank the midline in two parallel lines A and 3C.wt-GFP and ZO-1-mCherry in live Tg(HSP70:N-Cadwt-GFP)pt137/Tg(HSP70: ZO-1.1-mCherry)pt117b double transgenic embryos. We found that the foci of both proteins generally moved in the basal-to-apical direction—although they often traveled basally briefly before moving apically again—and fused into the PAAs and OAAs by 14-ss; in addition, their apical enrichments occurred earlier in the anterior regions than in the more posterior regions , the initial targeting of newly synthesized N-Cad to the basolateral membranes, translocation of N-Cad from the lateral membranes to the PAAs, and the likely blockade of N-Cadure PAAs I.At the level of subcellular adhesions, whereas the OAAs dissolve during the transition from the early neural rod to the late neural rod, the PAAs persist. It is during this transition when intermediate proteins localize to the apical regions. Thus do intermediate proteins maintain the PAAs?nok mutants, the normal circumferential PAAs were lost and residual adhesions clustered to a corner of neighboring cells  sequentially enrich or localize to the apical regions to regulate apical cell-cell adhesions for proper neurulation  to ensure that the same neural tissue region was examined, we used the adjacent otic vesicle as a landmark; (2) to relieve the interferences from developmental variations among embryos, we visualized two or more proteins simultaneously in the same embryos to reveal the relative order of their localizations; and (3) to reveal proteins' subcellular localization, we relied mainly on immunohistochemistry of endogenous proteins and used transgenic fluorescent fusion proteins as auxiliary and confirming measures. With these precautions, we revealed that pioneer proteins , intermediate proteins , and terminal proteins  and OAAs (AJ-like), thus shifting the cell-cell adhesion balance toward the apical ends , and S4.nokm520 mutation causes more severe defects than pard6γbfh266 mutation, possibly due to the hypomorphy of pard6γbfh266 mutation or pard6 gene redundancy , a symmetric collective , and opposing configuration . Here asymmetry and symmetry represent opposite concepts, and yet they depend on each other for their very existence; the interdependency and yet opposition between asymmetry and symmetry is reconciled through apical adhesions, which on the one hand, establish the asymmetry of individual cells, and on the other hand, configure asymmetric individual cells into a symmetric collective.  Such a reconciling process needs to be quantitatively regulated on the levels of both time and space by following the three-step order of expression and localization of polarity proteins. If this stepwise order is not followed, severe disruption of neurulation may occur. For example, precocious expression of Lin7c by mRNA injection resulted in multiple neural tubes in treated embryos .The intertwining relationship between symmetry and asymmetry goes beyond tissue formation and can be applied universally at hierarchical levels: all symmetric entities are collectives that are composed of opposingly configured asymmetric components, which themselves display a lower level symmetry, whether perfect or not G. Also, Our study reveals a hierarchical framework for zebrafish neurulation: pioneer, intermediate, and terminal polarity proteins enrich and localize apically in a three-step order to orchestrate the formation of a mirror-symmetric neural rod and then a neural tube by dynamically regulating the PAAs and OAAs. This framework implies a general principle that may govern the genesis of many mirror-symmetric tissues: tissue symmetry can be established by organizing asymmetric cells in opposing configurations via polarized adhesions, just like a magician's double-headed coin can be made by gluing two coins together tail to tail. By this simple and yet robust mechanism, the yin-yang duality of tissue symmetry and cellular asymmetry can be reconciled to become an inseparable oneness—asymmetry constructs symmetry and symmetry accommodates asymmetry.All methods can be found in the accompanying


Lactosomes accumulate in tumors in vivo through the enhanced permeability and retention (EPR) effect, even on frequently administering them. However, lactosomes cannot be efficiently internalized by cells. To improve cellular uptake of lactosomes, cell-penetrating peptide (CPP)-modified lactosomes were prepared. Seven CPPs (including EB1 and Pep1) were used, and most of them improved the cellular uptake efficiency of lactosomes. In particular, EB1- and Pep1-modified lactosomes were efficiently internalized by cells. In addition, by using CPP-modified and photosensitizer-loaded lactosomes, we demonstrated the photoinduced killing of mammalian cells, including human cancer cells. Accumulation of the EB1-modified lactosomes in NCI-N87 tumors was shown by in vivo imaging. Thus, this study demonstrated that the CPP-modified lactosome is a promising drug carrier.Polymeric micelles that are composed of synthetic polymers are generally size controllable and can be easily modified for various applications. Lactosomes (A  The use of nanoparticles as drug carriers for the selective delivery of pharmacological agents in various diseases such as cancer and viral infections has been studied. Over thel-lactic acid) (PLLA) block (AB-type)..No potential conflict of interest was reported by the authors.


Angiogenesis is essential for tumor growth and metastasis. Understanding the regulation of tumor angiogenesis has become increasingly important. MicroRNAs (miRNAs) are small noncoding RNAs that function in diverse biological processes via post-transcriptional regulation. Extensive studies have revealed two important regulatory roles of miRNAs in tumor angiogenesis: miRNAs in tumor cells affect the activity of endothelial cells via non-cell-autonomous mechanisms, and miRNAs in endothelial cells regulate the cell-autonomous behavior. Recent advances have further highlighted the role of tumor-derived extracellular vesicles in the regulation of tumor angiogenesis via transferring miRNAs to endothelial cells. In this review, we summarize the regulatory role of miRNA in tumor angiogenesis, with a highlight on clinical implications of miRNAs as biomarkers for anti-angiogenic therapy response, and as therapeutic interventions against tumor angiogenesis in vivo. Inducing angiogenesis is one of the hallmarks of cancer . Tumor-aAngiogenesis is a complex process by which new blood vessels are formed from pre-existing ones by sprouting, remodeling and expansion of primary vascular networks . In normMicroRNAs (miRNAs) are small non-coding RNAs , that neIn our review, we will summarize the non-cell-autonomous and cell-autonomous roles of miRNAs, as well as EV-transferred miRNAs in the regulation of tumor angiogenesis. Moreover, we highlight the clinical implications of miRNAs as predictive biomarkers for anti-angiogenic therapy response in both tissue and circulation, and several miRNA delivery approaches that have been applied to suppress angiogenesis in vivo.Angiogenesis is under the control of pro-angiogenic and anti-angiogenic factors . Tumor cThe most predominant growth factor of angiogenesis is VEGF, consisting of VEGF-A/B/C/D and placental growth factor (PlGF) , 17. VEGMiR-20 , miR-29bApart from directly targeting VEGF, a handful of miRNAs regulate VEGF-dependent tumor angiogenesis by targeting VEGF inducers, such as HIF pathway , HDGF (TSP-1) are endogenous anti-angiogenic factors [Angiogenesis is regulated by the balance between pro- and anti-angiogenic factors. Angiostatin, endostatin and thrombospondin-1  are primarily located on the surface of ECs , 81. TheMiRNAs have been implicated as key modulators of angiogenic signal transduction pathways. MiR-125b suppressed tube formation of ECs by inhibiting VE-cadherin translation . MiR-34aThe discovery of extracellular vesicles (EVs) represents an exciting area of research that offers novel mechanisms for intercellular communication. EVs, generally including exosomes  and microparticles or microvesicles , are small secretory vesicles that contain proteins, DNA, and coding and non-coding RNAs . EVs canRecent evidence has demonstrated that cancer cell-derived EVs can be transferred to ECs where they induce pro-angiogenic effects. Glioblastoma cells released exosomes containing mRNA, miRNAs and angiogenic proteins. These exosomes were taken up by brain microvascular endothelial cells and modulated angiogenesis . A set oMiRNAs are the most abundant non-coding RNAs enriched in EVs, and studies have also showed that EVs contain components of the RNA-induced silencing complex (RISC), suggesting potent regulatory role of EV-miRNA in recipient cells . SeveralHypoxia is a potent trigger of angiogenesis. Some studies have focused on clarifying how hypoxia affects tumor angiogenesis through tumor cell-derived EVs. Hsu et al. found that more exosomes were produced under hypoxic conditions than normoxic conditions by lung cancer cells. Tumor-derived exosomal miR-23a directly suppressed the expression of prolyl- hydroxylase 1 and 2 (PHD1 and 2), leading to the accumulation of HIF-1α in ECs and increased angiogenesis . TadokorTumor-induced vascular vessels commonly have abnormal structure with increased permeability and incomplete cellular junction . Zhou etAs angiogenesis is essential for tumor growth and progression, anti-angiogenic therapies have been developed to improve the outcomes of cancer patients. Several agents, including bevacizumab, aflibercept and most recently ramucirumab that target the VEGF/VEGFR signaling pathway, have been approved across some cancer types. In addition, multi-targeted tyrosine kinase inhibitors (TKIs), such as sorafenib and sunitinib, also block angiogenesis signaling. However, the overall response rate of these anti-angiogenic therapies is unsatisfactory in the clinic. Therefore, selecting out the patients, who would benefit from anti-angiogenic treatment, will help to promote therapy efficacy and avoid unnecessary toxic adverse effect. Due to the high tissue specificity and stability of miRNAs and their altered expression in tumor angiogenesis, miRNAs have been suggested as potent predictive biomarkers for therapeutic response Table .Table 4Tp = 0.04). Multivariate analysis revealed that miR-378 expression was an independent predictor for PFS after anti-angiogenic treatment [Bevacizumab, a humanized monoclonal antibody against VEGF-A, has been approved as combination therapy or monotherapy for first- and second-line treatment of several advanced cancers, including metastatic colorectal cancer (mCRC), metastatic renal cell carcinoma (mRCC), metastatic non-small cell lung cancer (NSCLC), progressive glioblastoma, metastatic cervical cancer and ovarian cancer etc. . In TCGAreatment . For patreatment .Several multi-target TKIs have potent anti-angiogenic effects due to the inhibition of VEGFR and PDGFR, and have been approved, including sorafenib for mRCC and unresectable hepatocellular carcinoma (HCC), and sunitinib for mRCC. High level of miR-425-3p was associated with longer PFS in HCC patients treated with sorafenib. Multivariate analysis confirmed the predictive significance of miR-425-3p, suggesting that assessment of miR-425-3p levels in liver biopsies could help in stratifying patients with advanced HCC for sorafenib treatment . Khella Although tissue miRNA expression could serve as molecular classifier for clinical outcome, there are several drawbacks including small quantities of diagnostic tumor tissue, genetic tumor heterogeneity in space and time, and unavailable acquisition of repeated biopsies. Therefore, circulating biomarkers offer a non-invasive opportunity for early diagnosis, prognosis evaluation and drug response prediction in cancer. Circulating miRNAs are stable and reproducible in blood due to their incorporation in exosomes/microvesicles or protein complexes, protecting them from degradation by endogenous RNase , 110. TeHansen et al. investigated the predictive value of circulating miR-126 in mCRC patients receiving first-line chemotherapy combined with bevacizumab. Blood samples of patients were collected before treatment (baseline), 3 weeks after treatment and at progression. Evaluation of miR-126 expression in plasma showed that non-responders had an increase in miR-126 level relative to baseline, while responders with decreased miR-126 alteration. The results indicated that changes in circulating miR-126 during treatment represented a possible biomarker for the response to anti-angiogenic containing therapy . M. JoerGiven the important regulatory role of miRNAs in tumor angiogenesis, targeting or delivering miRNAs to tumors holds great promise as a novel therapeutic approach. In this case, it is essential to deliver antagomiRs or miRNA mimics into tumor cells or endothelial cells. As naked miRNAs are hydrophilicity, unable to pass through cell membranes, and could be easily degraded by serum RNase , in vivoNanoparticle delivery systems, including viral and non-viral ones, have been addressed to protect miRNAs from degradation by serum nucleases when administered systemically . The advTo turn miRNA into therapeutics, it is critical to deliver antagomiRs or miRNA mimics specifically into target cells. Endothelial cells in the tumor microenvironment have been actively targeted in vivo for miRNA delivery. The cyclic Arginine-Glycine-Aspartic acid (cRGD) peptide coupled nanoparticle is most widely used. The cRGD could bind to integrin αvβ3 and αvβ5, which are present on tumor ECs, as well as on certain tumor cells. Studies have reported that systemic administration of anti-miR-296, anti-miRNA-132 or miR-7 mimics using the cRGD modified nanoparticles strongly reduced angiogenesis and tumor burden in mice, offering promise for miRNA-based anti-tumor therapeutic , 120. InCell-derived membrane vesicles (CMVs), including exosomes and microvesicles, are endogenous carriers transporting biological molecules to recipient cells. In contrast to established synthetic nanoparticles, CMVs are natural transporters and hence, less likely to exert toxicity or immune response, which probably due to their lowhoming to the liver , 124. MoZhang et al. revealed that CMVs derived from HEK293T cells are capable of packing and transporting miR-29a/c into gastric cancer cells. The in vivo experiments illustrated that CMVs could stably transfer miR-29a/c into the implanted tumor cells in mice, playing anti- angiogenic role by directly targeting VEGF . Their dSignificant advances have been made in exploring the regulatory role of miRNAs in tumor angiogenesis. The rapidly increasing discoveries shall pave the way in the use of miRNAs as predictive biomarkers for anti-angiogenic treatments and as miRNA-based strategy against tumor angiogenesis in the future, though there are some challenges.Given the low response rate of anti-angiogenic agents, a step forward would be discovering predictive biomarkers to distinguish responders from non-responders. So far, several candidate miRNA biomarkers have been identified, but they emerged from small studies and should be confirmed in large prospective trials. Besides, there has been no unified control miRNAs for normalization in the analysis of circulating miRNA expression. A proposed solution to this problem may be the introduction of exogenous control .Multi-targeted anti-angiogenic approach might exert increasing antitumor efficacy. Since a single miRNA has the potential to regulate angiogenesis by targeting multiple mRNAs, miRNA holds great promise as therapeutic approach for tumor angiogenesis treatment . HoweverAt the same time, developing technologies to deliver miRNAs to specific cells in vivo is highly essential for their therapeutic application. Despite having many strategies reported, the successful delivery approach in vivo is still limited. Clinical translation has been hampered by dose-dependent toxicity upon systemic administration , 134. InIn summary, better understanding of the regulatory network of miRNAs in tumor angiogenesis provides new insights in the biological process of tumor-associated neovasculature. Advances in profiling and delivery system offer clinical translation potential of miRNAs as predictive biomarkers for anti-angiogenic therapy response and therapeutics against tumor angiogenesis.


The US FDA granted accelerated approval to pembrolizumab for microsatellite instability-high and mismatch repair deficient cancers. The response of programmed death-1 blockade in mismatch repair proficiency (pMMR) colorectal cancer is very poor, however, whether such treatment is effective in pMMR nasopharyngeal carcinoma (NPC) remains unknown.We report a case of a 51-year-old man with NPC. PET-CT scan revealed a space-occupying lesion in the left lung, and the pathologic result confirmed the occupying lesion originated from NPC. Meanwhile, both immunohistochemistry and PCR revealed that the occupying lesion belonged to pMMR NPC. The lung lesions largely shrunk after chemoradiotherapy. One year later, MRI showed brain occupancy, and brain lesion resection surgery was performed subsequently. The resected tissue was also validated to be the metastatic lesion from NPC. After one month, the patient was examined again by PET-CT, which showed multiple metastases in the liver, pelvis and adrenal gland. Since January 2017, the patient has been treated with pembrolizumab therapy. After five courses of treatment, both PET-CT and blood testing were repeated and demonstrated that metastases and serum Epstein-Barr virus DNA almost completely disappeared.We provide the first report that pembrolizumab has a confirmed objective response to microsatellite stability and pMMR NPC, and two biomarkers may not be sufficient to identify patients who might be resistant to such treatment in NPC. Although various radiotherapy options have a high cure rate in NPC, recurrence and distant metastasis remain key challenges . TherefoPembrolizumab is the first anti-PD-1 antibody approved by the US FDA . A studyWe report the case of a 51-year-old man with MSS and pMMR NPC, who showed a “super response” to pembrolizumab treatment.A 51-year-old Asian male experienced rhinorrhoea with blood in 2012. He was given a clinical diagnosis of nasopharyngeal non-keratotic undifferentiated carcinoma  through nasopharyngoscope and pathological examination. The patient received concurrent chemoradiation and was followed up regularly at our hospital.In November 2015, a PET-CT scan indicated left lung-occupying lesion, which pathology confirmed originated from NPC. He was given nimotuzumab weekly and docetaxel in combination with nedaplatin 3 weeks for 6 cycles. After the chemotherapy, repeat PET-CT scan revealed the lesion had shrunk, then he received radiotherapy (50Gy/15F) for left lung peripheral metastatic disease. After the left lung peripheral lesions disappeared, he received further radiotherapy (DT 60Gy / 30F) for the left hilar residual because of hoarseness.In October 2016, MR scan showed brain occupancy  The patient had taken radiotherapy  before using anti-PD-1 treatment, especially high-dose radiotherapy for pelvic metastases at 5Gy. Ionizing radiation can induce DNA damage and cell death. The dead malignant cells will release tumour-associated antigens and damage-associated molecular patterns (DAMPs) such as high mobility group box 1, nucleotides, or heat shock proteins that can produce an immunogenic response , 16. ThuIn conclusion, our case report indicates that MSS and pMMR may not reflect resistance to anti-PD-1 therapy in NPC, high-dose radiation therapy before treatment and the expression of PD-L1 in tumour cells may contribute to predicting the response to such treatment. Further research is warranted to explore more precise biomarkers for different tumours.


Here, we report in vivo data for OTX015 in combination with the histone deacetylase inhibitor vorinostat, the Bruton's tyrosine kinase inhibitor ibrutinib, the anti-CD20 monoclonal antibody rituximab, and the mTOR inhibitor everolimus in a diffuse large B cell lymphoma model. The antitumor effect of OTX015-containing combinations in SU-DHL-2 xenografts in mice was much stronger than the activity of the corresponding single agents with almost complete tumor eradication for all four combinations. Pharmacokinetic analyses showed similar OTX015 levels in plasma and tumor samples of approximately 1.5 μM, which is equivalent to the concentration showing strong in vitro activity. For all four combinations, mean terminal levels of the bromodomain inhibitor differed from those in mice exposed to single agent OTX015, indicating a need for thorough pharmacokinetic investigations in phase I combination studies. In conclusion, our results provide a strong rationale to explore OTX015-containing combinations in the clinical lymphoma setting.The bromodomain inhibitor OTX015 (MK-8628) has shown anti-lymphoma activity as a single agent in both the preclinical and clinical settings, as well as  Diffuse large B cell lymphoma (DLBCL) is the most common lymphoma in Western countries and, despite improvements obtained with chemoimmunotherapy, up to half of these patients cannot be cured , 2. CurrAnalogous to the vast majority of human tumors and independent of their cell of origin, DLBCL cells also bear recurrent somatic mutations in genes coding for proteins involved in chromatin structure and remodeling that cause profound changes at the epigenetic level , 7. Of cin vitro and in vivo antitumor activity as a single agent in different lymphoma models, including ABC-DLBCL [in vitro synergism when combined with different agents in lymphoma models [in vivo activity of OTX015-containing combinations in an ABC-DLCBL xenograft model.OTX015 has BC-DLBCL . ClinicaBC-DLBCL , 26. AltBC-DLBCL –14. Sincin vitro synergism observed for combinations of OTX015 with other compounds [in vivo model of ABC-DLBCL. Mice bearing xenografts of the ABC-DLBCL cell line SU-DHL-2 were treated with control or OTX015, BTK inhibitor ibrutinib, the mechanistic target of rapamycin (mTOR) inhibitor everolimus, the histone deacetylase inhibitor vorinostat, or the anti-CD20 monoclonal antibody rituximab as single agents or in OTX015-containing combinations. None of the mice showed any body weight loss during the treatment period. When given as single agents, OTX015 and all four other drugs caused tumor growth delay  mice  were subcutaneously engrafted with 15 x106 cells of the human ABC-DLBCL cell line SU-DHL-2, and randomly divided into 10 groups of six mice each. Treatment with OTX015  was initiated three days after the engraftment, while treatment with ibrutinib , everolimus , vorinostat  or rituximab  was initiated when mice developed palpable tumors (100 mm3). Tumor size was measured twice weekly using a digital caliper. Tumor volumes were calculated as previously described [NOD-Scid , and stored at −80°C. Plasma concentrations were measured using a validated Ultra Performance Liquid Chromatography with tandem Mass Spectrometry method, as previously described . For tis