Preclinical studies demonstrate that a broad spectrum of human malignant cells can be killed by oncolytic paramyxoviruses, which include cells of ecto-, endo-, and mesodermal origin. In clinical trials, significant reduction in size or even complete elimination of primary tumors and established metastases are reported. Different routes of viral administration , and single- versus multiple-dose administration schemes have been explored. The reported side effects are grade 1 and 2, with the most common among them being mild fever. Some advantages in using paramyxoviruses as oncolytic agents versus representatives of other viral families exist. The cytoplasmic replication results in a lack of host genome integration and recombination, which makes paramyxoviruses safer and more attractive candidates for widely used therapeutic oncolysis in comparison with retroviruses or some DNA viruses. The list of oncolytic paramyxovirus representatives includes attenuated measles virus (MV), mumps virus (MuV), low pathogenic Newcastle disease (NDV), and Sendai (SeV) viruses. Metastatic cancer cells frequently overexpress on their surface some molecules that can serve as receptors for MV, MuV, NDV, and SeV. This promotes specific viral attachment to the malignant cell, which is frequently followed by specific viral replication. The paramyxoviruses are capable of inducing efficient syncytium-mediated lyses of cancer cells and elicit strong immunomodulatory effects that dramatically enforce anticancer immune surveillance. In general, preclinical studies and phase 1–3 clinical trials yield very encouraging results and warrant continued research of oncolytic paramyxoviruses as a particularly valuable addition to the existing panel of cancer-fighting approaches. Viruses can be quickly modified by recombinant DNA technology thereby rapidly incorporating the fast growing knowledge into oncolytic virus design. The studies involve a wide array of virus species belonging to diverse viral families, such as adenoviruses, herpesviruses, parvoviruses, enteroviruses, reoviruses, rhabdoviruses, paramyxoviruses, myxoviruses, alphaviruses, and poxviruses. Examples of clinical trials include a phase 2 trial of reovirus in combination with chemotherapy in patients with head and neck cancer8 and a phase 2 trial of genetically engineered oncolytic poxvirus JX-594 in patients with hepatocellular carcinoma.9 These trials confirm that oncolytic viruses do not produce substantial side effects and have considerable antitumor efficacy that affects the overall patient survival.The idea of using viruses in the treatment of malignancies dates back to the beginning of the 20th century when reports on cases of spontaneous tumor regression after viral diseases or vaccination started to appear.10Recently, the first oncolytic virus treatment in the United States was authorized by the Federal Drug Administration. Government agency approval was granted after completion of phase 1–3 trials that enrolled metastatic melanoma patients. The first oncolytic virus approved by the Federal Drug Administration is a herpes simplex virus-1 construct that encodes human granulocyte-macrophage colony-stimulating factor (GM-CSF).Innate barriers against viral infection exist at the molecular, cellular, tissue, and organism levels. Ordered tissue organization provides strong protection against virus penetration and spreading. In contrast, cancer cells form disordered structures, which are virus accessible. They lose their duties in the organism and form a separate foreign tissue (tumor) with the sole selfish function of accelerated expansion. The transition makes cells of a tumor a preferable substrate for oncolytic viruses in general and paramyxoviruses in particular .26–30 Functional p53 is commonly lost through different mechanisms in cancers32 and therefore malignant cells frequently do not commit suicide in response to viral infection and efficiently complete virus production.Many viruses have developed mechanisms that modify biosynthetic processes in quiescent cells to stimulate viral replication. Cancer cells already have high metabolic rate and therefore efficiently support propagation of viruses even if they have defects in the host-modifying functions. In response to viral infection, normal cells trigger p53-dependent suicidal programs that kill the abnormal (infected) cells before they start shedding viral progeny.Virus-infected normal cells start producing interferons (IFNs) that provide antiviral protection for surrounding cells and limit virus spreading. Cancer cells are usually not only defective for the induction of IFNs, but also do not develop resistance to viruses in response to IFN treatment.34 Loss of p53 functions in cancer cells voids the control mechanisms that ensure their social behavior: the p53-deficient cancer cells can be considered as separate organisms with very unstable genomes that enter the Darwinian competition/selection process. Along with mutations that promote accelerated proliferation and invasion, cancer cells get rid of functions that serve the needs of the whole organism. The induction of IFNs is compromised in multiple tumor types, in particular, through a homozygous loss of chromosome 9p21 region, which encodes type I INF genes.21In normal cells there is tight crosstalk between the IFN type 1 and p53 pathways. Acting in concert, these two pathways efficiently limit viral infection.35 The double-stranded RNAs elicit a strong type I IFN response that hinders proliferation of cancer cells thereby putting a strong selective pressure and forcing inactivation of the IFN response mechanisms so that further evolution and expansion of cancer cells35 is allowed. Cancer cells are generally better hosts for viruses as they acquire defects in the protective mechanisms that resist viral replication. The upregulation of p53 by type I IFNs plays a protective role against the emerging cancer cells.34 Therefore, cancer cells experience strong selection pressure against both the p53 and the type 1 IFN-mediated mechanisms. During the malignant progression, cancer cells acquire mutations in different components of the IFN system, p53 and apoptotic pathways36–39 that allow them to escape from the host regulation and to expand. But the very same defects that promote tumor growth provide the opportunity to destroy cancer cells with the use of oncolytic viruses. This also relates to paramyxoviruses.Frequent loss of type I IFN response in cancer cells is greatly stimulated by the impaired function of the p53 tumor suppressor. As a transcription regulator, p53 is responsible for silencing transcription from LINE and SINE transposons. Inactivation of p53 is associated with a dramatic activation of self-complementary transcripts (double-stranded RNAs) from the widespread repetitive DNA elements such as Alu-repeats.IFIT1. In the MV resistant cell lines, inhibition of viral replication was going along with strong expression of these molecules. In contrast, susceptible cell lines showed a much weaker expression of IFIT1. Pretreatment with IFN-β made the susceptible cell lines more resistant to MV-mediated oncolysis.40For example, the oncolytic potential of measles vaccine virus (MV) in a few sarcoma cell lines was negatively correlated with upregulation of expression of RIG-I molecule and the IFN-stimulated gene 41–43However, the preferable killing of cancer cells directly by a virus is not the only mechanism of viral oncolysis. Viruses are potent inducers of innate and adaptive immune responses that greatly contribute to the process.45 Introduction of a nonpathogenic virus strain stimulates natural immune surveillance mechanisms triggering the immune system to recognize and attack malignant cells.46Both innate and adaptive branches of immune system act against viral spreading and persistence in the organism, which in case of oncolytic virus therapy might impose a problem. However, host immune system also recognizes and attacks cancerous cells, and viruses can greatly stimulate the process. Preclinical and clinical data suggest that oncolytic viruses can be regarded as an efficient approach to cancer immunotherapy.47 While accumulated in tumors, pathogens bring into any tumor mass a lot of biological materials which is rich with foreign antigens, pathogenic RNA or DNA, and promote multiple danger signals with immuno-triggering properties. In other words, appearance of foreign proteins and genetic material in the malignant cells or near these cells in tumor masses could increase these cells visibility for the immune system.47 Numerous cytokines, including IFNs, induced by the pathogen and virus in particular, stimulate antigen processing in immunoproteasomes and presentation of cancer specific epitopes by major histocompatability complex molecules.48Many viruses, bacteria, and other pathogens have a tendency to accumulate in primary tumors and metastases.50 The abundant presence of sialoglycoproteins on the surface of cancer cells51 most likely promotes preferential association of the virus with malignant rather than with normal cells and contributes to their selective cytolytic effect in primary tumors and in metastases. UV-inactivated SeV is able to kill prostate carcinoma cells but unable to kill normal prostate epithelium cells. Perhaps, high sensitivity to SeV-mediated cell death of sialic acid–rich prostate carcinoma cells in comparison with normal prostate epithelium is explained by SeV’s preferential association with malignant cells.52Some oncolytic paramyxoviruses might have particular high affinity to cancer cells due to overexpression of viral receptors on their surface. NDV, mumps, and SeV use sialic acid–containing sialoglycoproteins as the cell surface receptors.53 MV Edmonston strain can kill cells that overexpress this receptor without significant cytopathic effect against nontransformed cells expressing low receptor levels.54 Nectin 4 has been identified as the additional receptor for MV.19 It is a member of adhesion receptors of the immunoglobulin super-family localized to the adherents’ junctions of epithelial cells. Nectin 4 can be considered as a tumor cell marker for breast,55 lung,16 and ovarian cancers,17 suggesting that it can be partially responsible for the selectivity of MV toward cancer cells.The attenuated measles virus (MV Edmonston strain) uses the CD46 receptor, which is a regulator of complement activation that is universally expressed in all nucleated human cells but is often overexpressed in tumor cells.56 analyzes these advantages in detail, while a brief description of some of the advantages is provided below.Are there any advantages in using paramyxoviruses versus representatives of other viral families as oncolytic agents? Recently published review58 Metastatic cancer cells often express a high density of sialic acid-rich glycoproteins that increase the invasive potential.13A potential unique advantage of some paramyxoviruses is the sialidase (neuraminidase) activity of their HN protein, which has the potential to remove sialic residues from the surface of tumor cells.59 The HN proteins present in SeV, NDV, and some other paramyxoviruses possess neuraminidase  activities.58 Neuraminidase is capable of cleaving and removing sialic acid residues from the surface of malignant cells leading to a dramatic increase in their ability to induce the T-cell response.60One of the possible mechanisms linking the increased sialylation with malignant phenotype is the creation of a thick “coat” on the cell surface that hides cancer antigens and provides an escape of malignant cells from the immunosurveillance. Removing some sialic acid residues from the surface of malignant cells by sialidase can unmask cancer specific antigens and make cells visible to the immune system. The removal of sialic acids from tumor cells is associated with a reduced growth potential, an activation of NK cells, and a secretion of IFN-γ.61 We believe that the ability to trigger syncytium formation is another great advantage of oncolytic paramyxoviruses. Syncytium formation is a mechanism of spreading infection without the release of mature virus particles from cells. Most likely this mechanism contributes to the efficiency of viral oncolysis because it allows extra rounds of viral replication without any viral exposure to host neutralizing antibodies.Some oncolytic viruses including paramyxoviruses are capable of inducing syncytia formation. During this process, infected and neighboring cells (up to 50 or even 100) are fusing with each other and are forming large multinucleated cells.63 Death of syncytia is associated with autophagy.64 A number of paramyxoviruses, including MV,65 NDV,66 and SeV,67 have been shown to induce autophagic programmed cell death. It was demonstrated that autophagy within the antigen donor cells facilitates antigen cross-priming to generate TAA-specific or virus-specific CD8+ T cells, which could be further explored in the future as a strategy to enhance oncolytic viruses-mediated antitumor effects.41 In addition, cross-presentation of TAAs by DCs is strongly promoted by paramyxoviral fusogenic membrane glycoproteins.63Moreover, syncytia are immunogenic formations; they secrete an abundance of “syncytiosomes,” which are exosome-like fluid filled cavities. The “syncytiosomes” present tumor-associated antigens (TAAs) through major histocompatability complex molecules.69 Perhaps, isolated immune therapy with UV-inactivated viruses, which could not kill cancer cells directly through infection, should be most efficient in patients with relatively a small tumor burden. This approach is exploiting the UV-inactivated virus as tumor-debulking immunotherapy. It is likely that another approach of using alive and cancer cell replication competent virus strain could exploit both virus direct oncolysis and tumor debulking immunotherapy and perhaps could deal with larger tumor burden. It engages the array of mechanisms that can contribute to favorable therapeutic outcome and give hope to patients with the most advanced and surgically non removable cancers.In summary, oncolytic paramyxoviruses are powerful anticancer immuno-stimulating agents. Strong anticancer effect of UV-inactivated viruses that cannot replicate or spread demonstrates most clearly the significance of the virus-induced anticancer immune response.Paramyxoviridae family) are associated with a number of diseases in animals and humans, such as MV, mumps, and several respiratory infections , human parainfluenza viruses, metapneumovirus, etc.). Canine distemper virus (CDV) and Rinderpest virus are associated with lethal infections in dogs and cattle, respectively. Sendai virus (SeV) affects mice and some other animals. Newcastle disease virus (NDV) is associated with a contagious disease affecting many domestic and wild species of birds. Few representatives of paramyxoviruses NDV, SeV, MV, and mumps viruses were tested as oncolytic agents in multiple model experiments and in a few clinical trials. These representatives are marked with a circle in a phylogenetic tree of Paramyxoviridae family  exhibit a low homologous recombination rate.75In general, because of a lack of a replicative proofreading mechanism, RNA viruses have higher mutation rates than those of double-strand DNA viruses.i.e., their genomes mainly including six genes, which encode for six major proteins. The unusual genomic requirement for polyhexameric length (6n+0) is likely responsible for particular low rate of homologous RNA recombination in paramyxoviruses.76 This low rate probably contributes to comparatively high viral genomic stability. Other explanations for this relatively high genomic stability of paramyxoviruses also exist. Perhaps, tight cotranscriptional wrapping of a viral ribonucleoprotein complex of paramyxoviruses prevents homologous recombination.Moreover, paramyxoviruses also belong to viruses that are governed by the “rule of six”, Natural high genomic stability of paramyxoviruses is a positive feature for their potential use in any anticancer clinical application. For such application, it is important that viral and foreign genes would be expressed from a viral genome in a comparatively stable way, so many serial passages in cell cultures or embryonated chicken eggs can occur without genomic change.77 It can be detected in animal colonies worldwide. While SeV spreads in rodents through both air and direct contact routes,77 it is considered quite safe for humans. Studies of the anticancer effects of SeV, which is also known as murine parainfluenza virus type 1 or hemagglutinating virus of Japan (HVJ), are mainly performed in Japan. SeV has oncolytic properties. Genetically engineered recombinant Sendai virus (rSeV) disseminates extensively in human tumor xenografts in nude mice without spreading to the surrounding normal cells.78 This dissemination leads to the inhibition of tumor growth in the mice. The tested tumor cells include fibrosarcoma, pancreatic epithelioid carcinoma, and human colon carcinoma.78 A significant reduction of tumor growth, including the complete elimination of established brain tumors, was demonstrated in murine models in a study using a different rSeV strain.79 Similar results were obtained with mouse xenografts of human sarcoma and prostate cancer.80 Recombinant SeV efficiently eliminated tumors in rat models, including melanoma, hepatocellular carcinoma, neuroblastoma, squamous cell carcinoma, and prostatic cancer.81 Some case studies that involved treatment of human cancers in a number of patients with SeV are reported in an issued patent82 and in patent application.83The SeV virus is responsible for a highly transmissible respiratory tract infection in mice, hamsters, guinea pigs, and rats.69 renal,69 and prostate carcinomas in mouse models.52 The UV-inactivated virus with enhanced antitumor activity was constructed by conjugation of IL-12 with hemagglutinin-neuraminidase (HN)-depleted viral particles (HVJ-E).84 It was demonstrated that this novel immune-stimulatory pseudovirion suppresses lung metastatic melanoma growth by regionally enhancing IFN-γ production without increasing the serum IFN-γ level.84Remarkably, the replication of SeV is not absolutely required for the anticancer effects of SeV as even UV-inactivated virus was shown to be efficient against colon,85In all mentioned studies, SeV eradicated the tumors or significantly inhibited their growth. The apparent safety of the virus and the promising preclinical data suggest that SeV could be an excellent candidate for oncolytic virotherapy. Supporting these hopes is a case of a short-term remission in a patient with acute leukemia following an intravenous injection of live SeV described back in 1964.Avulavirus genus, have been studied for almost half of a century since the pioneering work of Flanagan et al.86 that was performed with mouse Ehrlich carcinoma. The availability of attenuated strains of NDV that are used as live vaccine for controlling the disease in the poultry industry provides additional opportunity of studying oncolytic effects with minimal hazard for the environment.87 NDV is well-studied oncolytic paramyxovirus,89 and the approach of using NDV for cancer therapy has already moved into phase 1, 2, and 3 clinical trials.Oncolytic properties of avian NDV, which belongs to the 90 NDV strains also have been also classified into two categories lytic and nonlytic according to their ability of infecting monolayer tumor cells. So, lytic strains in these cells produce infectious particles that can infect other tumor cells, thus leading to an amplification of the viral load, while non-lytic strains produce noninfectious particles. Besides, infection of lytic NDV strains results in syncytium formation. The NDV strains that have been evaluated for the treatment of human malignancies are the lytic mesogenic derived strains MTH68/H, PV-701, and 73-T and the nonlytic lentogenic derived strain Ulster and HUJ (91NDV strains have been classified into three categories according to their virulence and pathogenicity to avian species: velogenic (highly pathogenic), mesogenic (moderately pathogenic), and lentogenic (low pathogenic). and HUJ .90,9193 A broad spectrum of human cancer cells were shown to be killed by NDV in vitro, which include tumor cell lines of ecto-, endo-, and mesodermal origin.94 Examples include the cells of colorectal, gastric, pancreatic, bladder, breast, ovarian, renal, lung, larynx, and cervical carcinomas, glioblastoma, melanoma,96 phaeochromocytoma,98 lymphomas of different origins,100 fibrosarcoma, osteosarcoma, neuroblastoma, and Wilms tumor.92 NDV has demonstrated a potent antitumor activity in several preclinical animal tumor models including neuroblastoma,101 fibrosarcoma,102 colon carcinomas,104 lung, breast, prostate carcinomas,103 hepatocellular carcinoma,105 and gastric carcinoma.106NDV was shown to kill human cancerous but not normal cells.95 because in normal cells viral infection rapidly induces production of a number of antiviral proteins.93In contrast, replication of NDV in nontumor cells could be up to 10,000-fold less efficient compared to some cancer cells,Morbillivirus) have been studied for more than a decade.107–109 The number of publications describing anticancer effects of attenuated measles vaccine virus strains is rapidly growing. The Edmonston B strain is considered to be very safe, as it has lost its pathogenicity after extensive passages in tissue culture and in chicken embryos.110 This strain has shown potent antitumor activity against multiple primary as well as established tumor lines and in several preclinical animal tumor models, including both solid tumors and hematology malignancies.61 Examples include lymphoma,111 multiple myeloma,112 medulloblastoma,113 glioblastomamultiforme,114 hepatocellular carcinoma,115 prostate,116 breast,118 and ovarian cancer.119 Different routes of viral administration , and single- versus multiple-dose administration schemes have been used in these models.61 The characteristic cytopathic effect involving the formation of multinucleated cell aggregates was observed in the treated animal tumors, followed by the apoptotic death of the infected tumor cells. The studies with oncolytic strains of MV are now moved to clinical trials.The anticancer effects of MV  was created. Expression of soluble marker (CEA) by the construct provided an opportunity for rapid, cost efficient assessment of viral replication in any organism .The ability to monitor viral gene expression in vivo imaging inspired the creation and use of another MV construct that encoded human sodium-iodine symporter gene (NIS). The NIS gene, expressed from a viral construct in tumor cells, is capable of promoting accumulation of radioactive iodine isotopes in these cells. This accumulation allows imaging of a tumor’s localized viral gene expression. Moreover, the accumulation also promotes specific radio-damage of malignant cells, providing an opportunity for radiotherapy in addition to viral oncolytic and immuno-stimulating effects of MV therapy. Both MV constructs MV-CEA and MV-NIS demonstrated efficacy and safety in preclinical models  was further explored by creation of another viral construct. The MV construct with an inserted extra gene of the prodrug-converting enzyme for 5-FC was designed to transform the nontoxic compound into a highly cytotoxic drug. It was shown that the MV construct with such a suicide gene is able to infect, replicate, and kill malignant cells taken from cancer patients. It was also shown that addition of the prodrug to the cells significantly enhances the cell killing. The prodrug-converting enzyme was extensively expressed in MV-infected cells from the tumor slices of the patient-derived materials.In vivo, intratumoral application of the MV suicide gene construct together with a systemic 5-FC treatment showed a significant reduction in tumor size in a xenograft mouse. The effect was stronger when compared with virus-only treatment.127 Intrahepatic and intraperitoneal applications of the same MV construct along with the systematic prodrug administration demonstrated comparative safety of the approach in animal models.128A number of case studies and clinical trials (mainly phase 1 and 2) were performed with oncolytic paramyxoviruses. The results are summarized in 129A phase 1/2a study for patients with advanced malignant melanoma was started recently in Osaka University (Japan).130 Moreover, antibodies to SeV and to human Parainfluenza virus type 1 (hPIV-1) are cross-reactive,130 so it is highly likely that the majority of adult humans that naturally have antibodies to hPIV-1 also have preexisting immunity towards SeV.SeV is a rodent pathogen. Administration of SeV to humans produces a very mild effect and does not cause any serious disease.7–108 embryo infective dose 50% of SeV every 7–10 days during 4 months. Virus was injected intradermally and occasionally intratumorally along with specific pathogen free chicken embryos disintegrated cells that include cells that are permissive for SeV replication and infectious virions production. The embryo cells coinjection allowed extra cycles of viral replication in patients’ skin. The viral therapy caused mild flu-like symptoms and in general was well tolerated. In Moscow trial 31 out of 47 patients responded to therapy, with 6 major responses . St Petersburg trail included patients with advanced metastatic cancers after surgery. Whole cell vaccines prepared from patients cancer cells were added to SeV formulations described for Moscow trial. The rationale for this addition is that tumor-specific antigens that are generally located in the plasma membrane of cancer cells may be better recognized by the immune system if they are associated with virus antigens. The cancer cells are treated with gamma radiation to leave cells alive but prevent further cell division. The trial included two groups. The first group incorporated 15 patients with variable malignancies that enter the trial after debulking surgery. Four patients in this group responded to therapy. They experienced remaining tumor shrinking and survived at least 1 year without disease progression. The second group included 12 patients with variable malignancies that entered the trial after radical cytoreductive surgery. Eleven patients in this group responded to therapy, they were observed to be disease free for at least 1 year. Unfortunately, all patients from St Petersburg trail were lost for follow-up after only 1 year of observation. Some case studies that involved treatment of human cancers in a number of patients with SeV are reported in an issued patent82 and in a patent application.83According to personal communication of Dr Senin, SeV (Moscow strain) was tested in a few groups of patients with advanced metastatic cancers (Russia). Moscow trial included a group of 47 patients with disseminated advanced disease for whom surgeries were not safe to perform and SeV was used as a monotherapy. The primary tumor localizations were variable. Patients were treated with one or two cycles of viral therapy per year with each cycle representing 10131 Administration of virulent strains of NDV to humans has been shown to result in only mild-to-moderate adverse effects, with mild conjunctivitis, laryngitis, and flu-like symptoms. Natural human infections with highly virulent avian NDV strains are possible, but they have been limited to mild conjunctivitis.132 The anticancer potential of NDV has been investigated in the United States, Canada, China, Germany, and Hungary. Several NDV strains  have been the subject of systematic clinical studies in patients who had exhausted all conventional cancer treatments.NDV is considered to be low pathogenic for humans while being highly contagious and epizootic in many domestic and wild avian species.per se,133–137 or its combination with oncolysates as well as whole cell vaccines prepared from cancer cells  and other components of immune system, however, degree of this stimulation could be variable. Unfortunately, so far, clinical studies do not provide clear cut answers as to which approach is most beneficial.There are at least two different, but not mutually exclusive conceptual applications of NDV and other oncolytic viruses. In theory, oncolytic virus could be used for direct tumor selective oncolysis or it could be used for triggering immuno-mediated cancer killing. Oncolytic virus could be injected as a sole agent or as a component of a tumor vaccine. So, clinical studies have evaluated the use of NDV er cells .138–142 144NDV therapy was tested using different routes of delivery . The studies have shown that the viral therapy causes mild flu-like symptoms and in general is well tolerated. It is lacks toxicity even at very high doses applied systemically.NDV-based anticancer therapy has been reported to be of benefit in more than a dozen clinical trials. However, the majority of the trials were comparatively small with less than a few dozen patients per trial. The efficiency of treatment was demonstrated for melanomas, glyoblastomas, head and neck squamous cell carcinomas, and some other malignant diseases.145 Among larger trials, two have to be mentioned. A phase 1 clinical trial with more than 100 patients suffering from variable advanced malignancies was performed in a few locations in Canada and in the United States. NDV was injected intravenously147 and demonstrated safety at high viral doses. It was noticed that some objective responses to the therapy occurred only at the highest dose levels. A phase 3 trial that involved 50 colorectal cancer patients was completed in Germany. It studied the efficiency of active specific immunization with NDV infected autologous tumor cell vaccine, following resection of liver metastases. In the total patient group, no significant difference in the disease-free survival or overall survival was detected between treated and control categories. However, in the subgroup of colon cancer patients improved 10-year overall survival was observed due to NDV therapy : 1.0–10.4; P = 0.042).142The mode of virus delivery seems to play a role in the efficiency of the cancer therapy. It was noted that intra- and peritumoral applications of NDV produce a stronger response compared to the systemic delivery, even when applied at much higher doses.148 is highly encouraging. Heinzerling et al. performed first phase 1 clinical trial of MV antitumor virotherapy using the Edmonston–Zagreb strain of MV on patients with cutaneous T-cell lymphoma. The study showed that intratumoral injection of MV induced local infection and characteristic cytopathogenic effect of virus on tumor cells, which was not abrogated by the presence of preexisting MV antibodies. Tumor regressions occurred in three out of five patients. Interestingly, some regression of distant lesions where MV was not injected was observed.149The overview of the early experience in ongoing clinical trials studies of MV constructs with patients suffering with ovarian cancer, glioblastoma multiforme, multiple myeloma, and cutaneous T-cell lymphomain vivo monitoring of viral gene expression in tumors of ovarian cancer patients that are negative for the CEA cancer antigen. The monitoring demonstrated that, after intraperitoneal delivery, the construct was able to infect patients’ tumor cells. De novo expression of CEA by these tumor cells was observed.150Addition of CEA antigen encoding sequence into the viral construct allowed cost efficient and quantitative 150Other interesting observations from the MV-CEA study include: (i) Anti-measles antibody titers in blood and in peritoneal fluid remain at constant level following multiple viral intraperitoneal deliveries. So, in other words, despite repeated viral administration, antibody titer boosting effects were not observed. (ii) Measles infection with wild type virus frequently leads to transient but strong immuno-suppression. No evidence of MV-CEA construct treatment-induced immuno-suppression was observed. (iii) There was no evidence of virus shedding in patients’ mouth gargle or urine samples. However, in a few patients, viral genomes were detected at low levels in the peripheral blood mononuclear cells.9 tissue culture infective dose 50% was observed. The median survival time of the virus-treated patients (more than 12 months) was double that of the median survival of the nontreated patient population (6 months). Side effects were grade 1 and 2, with the most common among them being mild fever, fatigue, and mild abdominal pain.150Most importantly, intraperitoneal treatment with MV-CEA of patients suffering from recurrent ovarian cancer revealed dose-dependent disease stabilization in the majority of patients (14 of 21). No dose-limiting toxicity with intraperitoneal infusion of viral material with a dose escalation of up to 10in vivo imaging of viral replication in malignant cells. This imaging became possible due to the ability of the NIS gene product from the MV construct to promote accumulation of radioactive iodine isotopes in tumor cells. The approach of in vivo visualization of MV-NIS replication was tested in preclinical studies (reviewed in refs. Addition of the human sodium-iodine symporter gene (NIS) into the MV construct, allowed 151Two relapsing drug-refractory myeloma patients with low pretreatment serum titers of antimeasles antibodies were treated by MV-NIS construct. After only one i.v. viral infusion, tumor-selective replication was observed. This tumor-selective viral replication led to complete remission of a disseminated malignancy in one patient. The remission lasted at least 9 months. All toxicities were comparatively mild and resolved within the first week after therapy.9 tissue culture infectious dose 50% was observed. Treatment was associated with a median overall survival of 26.5 months, which compared favorably with outcomes of therapies without the viral construct.152Another clinical study with the same viral construct and ovarian cancer patients demonstrated that no dose limiting toxicity with intraperitoneal infusion of the viral material with up to 10153 and wild type in a group of 90 patients with various malignancies at terminal stages154 as well as in a group of 200 patients with advanced cancers.155 The majority of the patients from these clinical trials experienced long-term suppression of tumor growth and certain oncolytic effects. For example, five of the seven patients with ascites experienced complete remission with no recurrence after intracavitary administration of the attenuated virus.153 Intravenous injections of the wild type mumps virus into 200 cancer patients caused a decrease or disappearance of ascites and edema of the lower limbs at high rates, a decrease or stoppage of cancerous bleeding and regression of tumors in 26 patients with cancer of the breast, rectum, thyroid gland, uterus, skin, etc.155 The published studies with mumps virus did not report median progression free or disease free survival times after therapy. Long-time survival benefit of treatment is unknown.A few studies have been carried out with wild-type and attenuated mumps virus. The attenuated virus was tested in a group of 22 patients with advanced gynecologic malignancies,Enhancing virus cancer cell killing abilities and diminishing bystander normal cells damage are modern day challenges for oncolytic virotherapy. These challenges trigger research that has already resulted in a number of interesting approaches that are waiting to be tested in clinical trials.in vivo in patients after systematic administration will define how broad its application is going to be.MV construct with an ability to transform the nontoxic compound into a highly cytotoxic drug (5-FC) was described above . So far, its efficiency was tested with the MV construct being administered locally. The ability of the construct to infect malignant cells 156 In the colon adenocarcinoma model, it was demonstrated that the MV construct significantly delayed tumor progression and prolonged median overall survival of treated animals. The effect of intratumoral application of the GM-CSF gene encoding MV construct compared favorably with the effect of MV virus without the inserted gene. Evidence was obtained that arming MV with GM-CSF improves the attraction of immune cells to a tumor and enhances the induction of a tumor-specific immune response.156 It is highly likely that the encouraging results obtained using the murine model can be translated into successful clinical trials benefiting cancer patients.Another approach for improving antitumor virus therapy efficiency involves insertion of the GM-CSF encoding gene into an MV genome.157 Another is programmed death-ligand 1 (PD-L1), which is a protein that also suppresses the immune system. Antibody to this protein was also successfully clinically tested as an anticancer agent.158 Using animal models and poorly immunogenic tumors, it was demonstrated that antibodies towards (CTLA-4) act synergistically with oncolytic virus. Preestablished distant tumors were efficiently rejected after local intratumoral NDV administration along with systematic CTLA-4 antibody administration. The effect was independent of tumor cell line sensitivity to NDV-mediated lysis.159The immunotherapeutic potential of oncolytic virus could be enhanced by coadministration of antibodies directed toward certain immuno-suppressive proteins. Two such proteins are of particular interest. One of them is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), which is a protein receptor that acts as an “off” switch when bound to some receptors on the surface of antigen presenting cells. Antibody to this receptor was already successfully clinically tested as an anticancer agent.160Another study with animal models showed that CTLA-4 and PD-L1 checkpoint blockade enhances oncolytic effect of MV. The effect was demonstrated by using a MV construct with extra genes that encoded CTLA-4 and PD-L1 antibodies. It was confirmed by coadministration of MV vectors along with CTLA-4 and PD-L1 antibodies.161 This work shows that expression levels of miRNAs in normal tissues can be used for adjusting virus oncotropism without compromising its oncolytic efficacy.Differential expression of miRNAs in normal versus malignant cells can be explored to increase oncolytic virus specificity, which improves its safety after systematic administration. Synthetic target sites for miRNAs that are overexpressed in liver, gastrointestinal tract, and other organs were inserted into the MV genome. It was demonstrated that a replication of such MV construct was repressed in nontransformed primary human hepatocytes and in liver slices. So, the virus replication was affected in cells expressing relevant miRNAs.25 For these viruses, a proteolytic processing enzyme is responsible for cleavage of precursor fusion glycoprotein F0 into fully active fusion protein dimer F1/F2. The functioning dimer protein is crucially important for the virions infectivity and syncytium formation. F0 is unable to fuse viral and cellular membranes for enabling viral penetration; moreover, it cannot direct fusion of infected cells with adjoining cells for syncytia formation. So, the malignant cell that does not express proteolytic processing enzymes either produces noninfectious defective virions or does not produce them at all.Paramyxoviral replication cycle requires protease cleavage of viral glycoproteins.0 processing, which is frequently overexpressed in metastatic cancers.162 Furin is a member the proprotein convertase (PC) family, processes inactive precursor proteins to functional proteins. Furin and other PC family members (furin/PCs) activate a number of proteins, including matrix metalloproteases (MMPs).163 MMP are specifically overexpressed in many metastatic cancers.24 The metastatic cells frequently secrete MMP, which is able to degrade the extracellular matrix and help these cells to metastasize. So, the expression and activity of furin as a proprotein convertase is necessary for processing of the enzymes, which stimulate tumor progression and metastasis.163 In summary, furin plays a significant role in tumor progression, invasiveness, and metastasis, so cancer cells frequently overexpress this protein.164–168 This furin overexpression most likely contributes to MV oncospecificity.The evidence exists that cancer specifically overexpressed proteinases could finalize the oncolytic cycle of paramyxoviruses. For example, MV virus requires proteinase-convertase furin for F162 However, in so far as furin is activating MMP activity, it is not known if cancer cell’s MMP proteolytic activity is possible without furin being expressed.However, not all cancer cells express furin. So, the cancer cells that do not express this protein most likely are more resistant to MV oncolysis versus those cells that do express furin. Furin nonexpressing cells cannot produce infectious MV particles, which promote intratumoral syncytia formation. The introduction of the multi-basic cleavage site into the F-protein allows this protein to be cleaved and to be activated by a broader range of proteases. In place of a gene encoded furin-activated F-protein, a gene encoded MMP activated F-protein was inserted. The recombinant virus infection was spread only in cells secreting MMP. So, this recombinant MV obtained ability to infect, destroy, and spread in tumors expressing MMP.169–171 Similar results were obtained when the F protein of the NDV La Sota strain was modified in an analogous fashion.173 “This improvement in syncytia formation caused a significant 20% prolongation of survival”.174To increase oncolytic potency of one lentogenic NDV strain, a polybasic cleavage site was also introduced into the F protein to generate a new site. While the resultant virus exhibited only an intermediate virulence phenotype based on a mean death time in embryonated eggs, the virus formed large syncytia and was enhanced in its replication in cancer cells, leading to enhanced oncolytic effects in various animal tumor models.176 Recombinant F protein of SeV enables its continuous spread in malignant human tissues without evidence of biodistribution into nonmalignant tissues.177The studies with SeV also demonstrated that introduction of specific cleavage site into the F protein allows utilization of broad range of tumor tissue-specific proteases.A number of factors define how well a particular patient will respond to oncolytic virus therapy. Among them are the patient’s immune status and a sensitivity of his malignant cells to a particular viral infection. It is highly desirable to develop cost efficient approaches for analyzing this sensitivity without testing virus infections in primary cultures from biopsy or surgical material. Investigation of individual’s malignant cells for their potential susceptibility to oncolytic virus infection and ability to produce infectious virions has to be fast and cheap.0–protein. Any malignant cell that is characterized by all three criteria mentioned above is more likely going to produce infectious virions and efficiently participate in intratumoral infection spread. The tumor, which consists of such cells, is likely vulnerable to oncolytic virus infection and consequent destruction.At least three factors contribute to development of permissive status by malignant cells that allows production of infectious paramyxovirus virions. First, a presence of a particular virus receptor, second, an absence of functioning IFN or/and apoptosis response pathways and, third, an expression of proteolytic processing enzymes for the F0–protein processing proteolytic enzymes.So, an investigation of this vulnerability is needed to detect the category of the patients that are going to respond to the oncolytic therapy. Consequently, it is highly desirable to test the malignant cells from the patients’ biopsy or surgical material for the presence, quantity of viral receptors, for status of IFN response mechanism and for expression of the FOverall, clinical study results are still preliminary, and the reported outcomes are very diverse. So far, it is difficult to compare the relative efficiencies of the different paramyxoviruses in clinical trials. Median progression-free survival, median disease-free survival time as well as hazard ratios of experimental versus well-matched control groups are frequently not reported. However, the studies demonstrate comparative safety of oncolytic paramyxoviruses and, frequently, some objective positive effects. This information is encouraging for the design of phase 2/3 trials.Future trials are likely going to be more successful because the factors that define how well a particular patient will respond to oncolytic virus therapy now are better understood. Consequently, better selection of the most likely responders among new patients becomes possible. Modern research demonstrated that the immunotherapeutic potential of oncolytic paramyxoviruses could be enhanced by coadministration of antibodies toward certain immuno-suppressive proteins. Moreover, the new sets of genetically modified viruses with enhanced virus cancer cell killing abilities as well as with optimized safety profiles are being made ready for clinical trials.


Traditional approaches of biomarker detection that consider only one aspect of the biological continuum like gene expression alone are limited in their scope and lack robustness in identifying the key regulators of the disease. We have adopted a multidimensional approach involving the cross-talk between the omics profiles  related to pancreatic ductal adenocarcinoma (PDAC). These domain-specific PDAC signatures were integrated to identify genes that were affected across multiple dimensions of omics space in PDAC . To further pin down the regulators of PDAC pathophysiology, a systems-level network was generated from knowledge-based interaction information applied to the above identified GMCs. Key regulators were identified from the GMC network based on network statistics and their functional importance was validated using gene set enrichment analysis and survival analysis.Multidimensional domain-specific disease signatures were obtained using rank-based meta-analysis of individual Rank-based meta-analysis identified 5391 genes, 109 miRNAs and 2081 methylation-sites significantly differentially expressed in PDAC . Bimodal integration of meta-analysis signatures revealed 1150 and 715 genes regulated by miRNAs and methylation, respectively. Further analysis identified 189 altered genes that are commonly regulated by miRNA and methylation, hence considered GMCs. Systems-level analysis of the scale-free GMCs network identified eight potential key regulator hubs, namely E2F3, HMGA2, RASA1, IRS1, NUAK1, ACTN1, SKI and DLL1, associated with important pathways driving cancer progression. Survival analysis on individual key regulators revealed that higher expression of IRS1 and DLL1 and lower expression of HMGA2, ACTN1 and SKI were associated with better survival probabilities.It is evident from the results that our hierarchical systems-level multidimensional analysis approach has been successful in isolating the converging regulatory modules and associated key regulatory molecules that are potential biomarkers for pancreatic cancer progression.The online version of this article (doi:10.1186/s13073-016-0282-3) contains supplementary material, which is available to authorized users. The generation of integrated system-level networks including the molecules dysregulated at regulatory , transcriptional (genes) and translational  omics levels helps in generating a complex network dysregulated in the disease. The analysis of this complex network can shed light on critical pathways and key molecules driving disease progression.Cancer is a complex disease that leads to dysregulation of multiple biological processes and pathways at regulatory, transcriptional and translational domains of central dogma. An extensive amount of genomics, proteomics and epigenetics data has been generated to probe the role of molecules from these domains in different pathways and biological processes involved in cancer pathophysiology. Most studies have concentrated on the individual contribution of molecules in disease pathophysiology, ignoring the interactions between Pancreatic cancer is a very aggressive form of cancer with poor diagnosis and dismal prognosis. Of the exocrine pancreatic cancers, 95 % are pancreatic ductal adenocarcinoma (PDAC), with increasing occurrence in the last decades . PatientAnother area that needs to be addressed in identification of biomarkers in PDAC is the basic biological processes affected in PDAC. It has been shown that dysregulation of multiple genomic spaces occurs in many cancers and the resultant disease is a culmination of multiple mutations; dysregulated control mechanisms, like regulatory microRNAs and epigenetic control; and dysregulated genes and proteins. The role of small non-coding microRNAs (miRNA) in regulating gene expression in cancer is being extensively studied. It has been shown the oncogenes and tumor suppressor genes are targets of differentially expressed miRNAs in solid tumors . Pederseomics studies and large compilations of multi-omics data like Gene Expression Omnibus (GEO) [omics dimensions. The complex nature of interactions leading to the pathophysiology of cancer warrants the use of systems biology-based approaches to understand the cause–effect relationship between the dysregulated molecules identified by traditional supervised analysis methods. Biological interaction networks provide insights into the biology that is fundamental to the disease process. Additionally, biological networks can help to identify key genes that are critical in pathophysiological cancer networks, thus leading to biomarker identification. These key genes (hubs) in the network regulate a plethora of important functional genes and their deletion, in many cases, results in lethality [omics domains can provide better insights about key regulatory (KR) molecules than individual dysregulated gene-based analysis, by taking into consideration the gene interactions [omics dimensions, namely, the transcriptome, regulatory miRNA and the epigenome, to build a systems-level network to identify the key molecules associated with PDAC. Further functional and survival analysis of the identified KR genes clearly depicted their association with pathways linked to cancer progression and survival. Additional file With the availability of data from different us (GEO) , the Intus (GEO)  and The us (GEO) , it is nethality . It has ractions . In thisRaw data on gene expression, miRNA expression and DNA methylation were obtained from public repositories, namely GEO and TCGA, and were normalized using R statistical software and Bioconductor packages in a platform-specific manner. Details about all the published datasets used in this study (accession numbers/references) can be found in Table The pancreatic cancer miRNA datasets used in this study are listed in Table The quality of the normalized data from Affymetrix gene expression datasets was assessed using the arrayQualityMetrics package from Bioconductor . IndividUnsupervised analysis was performed to further ascertain the quality of datasets and identify outliers without biological relevance, based on inter-array correlations using principal component analysis . PCA projects multivariate data objects onto a lower dimensional space while retaining as much of the original variance as possible , 21. PCAP-value < 0.05 were considered significantly differentially expressed in this conventional analysis approach. The list of differentially expressed molecules from each dataset was compared using Venn diagrams created using jvenn [We used the limma package  from theng jvenn  .Meta-analysis was performed to identify the differentially expressed molecules from multiple datasets using a rank-based approach  in Similar rank products-based meta-analysis was performed on multiple regulatory miRNA expression datasets and epigenome datasets to obtain PDAC meta-signatures of regulatory miRNAs and differential methylation, respectively. These gene, miRNA and methylation meta-signatures constitute the multidimensional PDAC signature used for further analysis.To determine the interactions between the epigenome and transcriptome, the genes and methylations sites were annotated using official HUGO gene symbol (version hg19 of the human genome). The interactions between methylation and gene expression signatures were deduced based on consensus genes found in both gene and methylation meta-signatures. These genes, which show differential expression as well as hypo/hypermethylation, are considered to be the genes regulated by methylation (methylation-regulated genes) irrespective of the directionality of gene expression or methylation. Biplots of gene expression changes with either methylation or miRNA expression changes were generated to extract any patterns between expression profiles  were considered the functional gene–miRNA interactions. Further, the genes that were regulated both by methylation and miRNA were considered genes under multiple regulatory controls (GMCs).The miRNA–gene interactions were determined using the RmiR package  from Biocs study . The difP-value using Fisher’s exact test for each pathway and functional category, according to the fit of the user’s data to IPA databases. The P-value measures how likely the observed association between a specific pathway/function and the dataset would be if it were due only to random chance. The functional categories and pathways with FDR < 0.05 were considered to be significantly associated.To identify biological pathways significantly overrepresented in miRNA-regulated and methylation-regulated genes, pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA) (Qiagen) software. The knowledge base of this software consists of functions, pathways and network models derived by systematically exploring the peer-reviewed scientific literature. It calculates the omics domains using Cytoscape 2.8, an open source platform for biomolecular network visualization [The meta-signatures of miRNA, genes and methylation were utilized in generating the network of interacting molecules from multiple lization . The genlization . The netlization . The genlization  plugin in is defined as the average connectivity of all neighbors of n [The network topological parameters were calculated using the CentiScaPe Plugin  in Cytosors of n . The AR Partitioning around medoids (PAM)  is a robP-values less than 25 % (FDR ≤ 0.25) were considered significant.Gene set enrichment analysis (GSEA)  was perfP-values from the log rank test were less than 0.075.To identify group-dependent patterns from the expression profiles of KR hubs identified using systems-level approaches, the self organizing map (SOM) clustering technique was adopted . SOM allomics data types) were integrated to generate a multidimensional PDAC signature associated with disease progression. The study included 177 gene expression profiles, 224 miRNA expression profiles and 210 DNA methylation profiles from different pancreatic cancer studies. Details about the datasets from which these profiles were obtained can be found in Table Pancreatic cancer data from multiple biological domains , cytoskeletal reorganization , kinetochore formation and spindle checkpoint , immune and inflammatory response , angiogenesis  and metalloproteinase . It was previously shown in a meta-analysis of PDAC expression data that many of these genes are indeed differentially expressed in PDAC [S100A11, ADAM8 and IL8.Rank-based meta-analysis using the rank products (RP) method was performed to address this inherent heterogeneity among datasets. RP performs a rank-based comparison of genes from different experiments to identify genes that are consistently ranked high (upregulated) and low (downregulated), clearly depicting advantages over linear modeling-based methods in meta-analysis by better handling noise from heterogeneous datasets. The RP-based method depicts a slightly better overlap among differentially expressed genes identified in meta-analysis . A heatmap of the top differentially expressed miRNAs obtained from meta-analysis is shown in Fig.  in PDAC . It was  in PDAC . Our resc cancer . miR-107bilities .CUX1, a member of the homeodomain family of DNA binding proteins, with a probable role in cell proliferation and cell cycle progression; CABLES1, involved in cell cycle regulation through interactions with cyclin-dependent kinases [RASA3, a Ras GTPase activator involved in the control of cellular proliferation; WIPF1, involved in cytoskeletal organization; STK17B, a serine/threonine kinase acting as a positive regulator of apoptosis; and TGFBR2, involved in multiple processes related to cell proliferation, differentiation, immunity and carcinogenesis in the cell. Many genes involved in specialized cellular functions were found to be hyper-methylated, for example, PRKCB, (protein kinase C), which plays an important role in B-cell activation; HOXD6 belonging to the homeobox family of genes; NTM, a neural cell adhesion molecule; and IGF2BP1 (insulin-like growth factor 2), an mRNA binding protein involved in translational regulation.Unsupervised analysis of normalized DNA methylation data depicted a significant separation between PDAC and normal samples along the top three PCs in PCA Fig. . Out of  kinases ; RASA3, The multidimensional PDAC signature consists of all significantly differentially expressed genes, miRNAs and methylation sites in PDAC identified from the meta-analyses (FDR ≤ 0.05) as described above.The meta-analysis of transcriptome, epigenome and regulatory miRNA expression identified a large list of differentially expressed molecules associated with PDAC. Even though the results provide a significant starting point for understanding disease pathophysiology, it is difficult to pin down disease-driving molecules from long lists of genes without considering the interaction between molecules. To obtain this integral cross-talk between the differentially expressed molecules, the bimodal interactions of genes with miRNAs and genes with methylation were first interpreted. The cross-talk between differentially expressed genes and miRNAs was deduced on the basis of sequence homology-based miRNA target gene prediction. Based on predicted gene–miRNA targets, 1150 genes were identified to interact with 49 miRNAs from the PDAC meta-gene signatures and, hence, expression of these genes is potentially regulated by miRNAs  Fig. .Fig. 4IdThe interaction between PDAC-associated gene expression and methylation changes was deciphered by comparing the differentially expressed and differentially methylated genes on the basis of HUGO gene symbols. Among the PDAC gene meta-signature, 715 genes showed overlap with the methylation meta-signature and were considered to be methylation-regulated genes Fig. .The inverse proportionality postulate between control mechanisms and gene expression was tested by generating biplots of gene expression changes with either methylation or miRNA expression changes  in breast cancers, leading to an aggressive breast cancer phenotype [miR-194 and miR-195, known to be involved in anti-tumor mechanisms in the cell, are downregulated in our PDAC signature and correlated with many of the identified GMCs in our study. For example, miR-194 is an epithelial marker and is known to target many genes involved in the epithelial-to-mesenchymal transition, thereby reversing the transition [miR-195 has been shown to induce apoptosis in human embryonic stem cells of neural origin [The results from functional gene–miRNA interaction analysis show that MCs Fig. . The rolexplored . Similarhenotype . The miRansition , whereasl origin .P-value) ≥ 10) functional categories revealed a considerable overlap between the two sets. Commonly affected functional categories include cellular growth and proliferation, cellular development, cellular movement, cell death and survival, organismal survival, organismal development, embryonic development, cancer, gastrointestinal disease, and cardiovascular system development  was the top enriched pathway with positive z-score indicating pathway activation. The TGFβ signaling pathway  was the most activated and the HGF signaling pathway  the most inhibited among the growth factor signaling pathways. It has been shown from analysis of TCGA RNA-seq data that TGF-β signaling is active in the PDAC transcriptome signature [PTEN signaling  was downregulated and was the top enriched pathway among apoptosis-related pathways. Among the cancer-related pathways, colorectal metastasis signaling  and PDAC signaling  were among the most enriched. Axonal guidance signaling and regulation of the epithelial–mesenchymal transition were the top enriched organismal growth and proliferation-related pathways. Multiple immune response-related pathways were enriched in CGMC network genes: IL-8 and leukocyte extravasation signaling pathways were activated whereas the CXCR4 signaling pathway was downregulated .The multidimensional PDAC meta-signatures were used to create a global network of 3386 nodes on the basis of available interactome information Fig. . To focuignature . PTEN siE2F3, HMGA2, RASA1, IRS1, NUAK1, ACTN1, SKI and DLL1. The top five hubs according to the AR score are shown in Fig. E2F3 is a top-ranking KR hub with 24 interacting neighbors that were significantly linked with G1/S transition of the mitotic cell cycle (FDR = 0.0021). The NUAK1 subnetwork consisted of 17 neighbors associated with the TLR signaling pathway (FDR = 0.00041). Previous studies have shown that tumor cells express functional TLRs and their activation leads to tumor cell proliferation and resistance to apoptosis [ACTN1 subnetwork had 22 interacting neighbors significantly associated with cell adhesion and cell junction organization (FDR = 8.57E − 05). The RASA1 hub had 34 interacting neighbors involved in many growth response signaling pathways like ERBB signaling (FDR = 4.71E − 11). SKI interacted with 13 neighboring nodes in the CGMC network and was involved in endocrine system development, pancreas development and negative regulation of TGFβ receptor signaling (FDR = 5.07E − 05).KR hubs were identified from the CGMC network through topological analysis on the basis of node interactions. The nodes were ranked based on neighborhood connectivity and node stress. From this analysis, eight KR hubs were identified (AR score ≤ 10), namely poptosis . The ACTP-value ≤ 0.05 and FDR ≤ 0.25)  and the DLL1 cluster had the highest negative enrichment score . ACTN1 and HMGA2 clusters showed a general overall distribution of genes in the network skewed more towards the upregulated genes. SKI showed a similar trend but skewed more towards the downregulated genes in the network. The results clearly show that even though we started with a set of differentially expressed genes that were not filtered based on fold change, the KRs and their subnetworks are formed from substantially differentially expressed genes. This subnetwork enrichment analysis has provided a way to understand the relative importance of the identified KRs in stabilizing PDAC network. The analysis also showed that IRS1, E2F3, DLL1, SKI, ACTN1 and HMGA2 are correlated with genes that are more differentially expressed in PDAC compared to RASA1 and NUAK1.The importance of the identified KRs in the CGMC network was assessed using a GSEA approach. The analysis was designed to understand the importance of subnetworks created from eight identified KRs in the overall CGMC network. The KRs were used as seeds to partition the CGMC network into PAM clusters using the k-medoids clustering methodology. This methodology identifies subnetworks on the basis of correlation of KRs with other genes in the network instead of just taking into consideration the first neighbors of KRs. The enrichment of these KR clusters in the CGMC network was assessed using GSEA. Six of the eight KR clusters showed statistically significant enrichment , indicating their prognostic role in PDAC.The significance of the KRs was further tested using survival analysis on the eight identified KRs in two PDAC expression datasets. The samples were partitioned into two groups by the most contrasting expression characteristics for the eight KRs using the SOM clustering approach, and survival analysis was performed on the two clusters  when samples were split on the basis of low or high expression of individual KRs . E2F3, which was the other KR that was significant in the GSEA-based subnetwork enrichment analysis, did not reach statistical significance in the individual survival analysis, but was able to discriminate poor survivors from better survivors to some extent. Thus 83 % of the KRs filtered through subnetwork enrichment analysis were also found to have significant prognostic value in the survival analysis, indicating that our systematic filtering approach has provided a valuable set of genes that are potential biomarkers with prognostic value in PDAC.To further ascertain the relative importance of these eigth identified KRs in survival, we performed survival analysis on individual KR genes. In the individual gene-based survival analysis, KRs Fig. . The anaomics data is the identification of the causal (KR) changes and differentiating them from bystander effects associated with the disease. Owing to the large number of genes measured in genomic assays, it is difficult to achieve statistical power except for the most highly differentially expressed molecules. In many cases the KRs remain unidentified owing to subtle changes at expression level. Even when identified as being differentially expressed, KRs are hard to characterize based on just their differential expression measure from a long list of altered molecules. However, the alternative approach of identifying regulatory genes from biochemical studies is laborious and time consuming and does not compare to genome-scale experiments in providing comprehensive coverage.One of the most challenging tasks in the analysis of large-scale Numerous studies have shown that network-based approaches that take into consideration the contribution of individual genes along with their interactions are an effective solution in identifying KRs of disease pathophysiology . It has Keeping this in mind, we adopted a multistep approach integrating the cross-talk between different genomics spaces  as well as an interactive network analysis to identify the key driver genes associated with PDAC. To achieve this, we initially performed rank-based meta-analysis individually on epigenetic, transcriptional and miRNA expression data to define a comprehensive landscape of molecular alterations in PDAC. It is well established that rank-based methods perform more reliably and consistently than non-parametric and parametric methods for meta-analysis of microarray data . In thisomics dimension  and is important for understanding the regulatory cascades and cross-talk between gene expression and upstream regulatory mechanisms. Understanding of the role of these epigenetic and post-transcriptional modifications in the development of disease phenotype could be crucial to identify regulatory bottlenecks that play causal roles in the disease. Profound epigenetic changes have been shown to associate with the onset and progression of cancer, and changes in DNA methylation is one of the potent underlying mechanisms [miR-21 and miR-222, which formed part of our multidimensional PDAC meta-signature, are both probable prognostic markers for PDAC [miR-222 was also identified as a KR of PDAC pathophysiology in our systems-level network analysis. In this respect, the interactive network analysis of miRNA subnetworks created from the global PDAC meta-signatures network reiterated the importance of the systems-level approach in understanding the cause–effect relationship between the dysregulated molecules (Additional file miRNA-221 and miRNA-222 were highly correlated and were at the center of a subnetwork created around miR-222 (Additional file miRNA-210 was one of the peripheral molecules in its subnetwork (Additional file miRNA-221 and miRNA-222 play a more causal role than miRNA-210 in PDAC disease progression. It was also evident from our analysis that miR-194, an epithelial marker that is downregulated in PDAC, is the center of the subnetwork created from miR-210.To generate a comprehensive view of alterations associated with PDAC as well as understanding the cross-talk among different genomic spaces, we adopted a multidimensional modeling-based approach. This step involved the integration of meta-analysis results from each chanisms , 59. Simchanisms , 61. In multi-omics studies with both gene expression and miRNA expression data on matched samples. Overall, in the multidimensional modeling step we identified 189 genes that manifested concerted changes across gene expression and their regulatory mechanisms in pancreatic cancer. As expected, this consensus multidimensional PDAC signature was enriched in important pathways associated with cancer progression, such as cell cycle regulation, cell growth and proliferation, cell motility, cell-based immunity, and inflammatory response.It has been postulated that dysregulation in regulatory mechanisms can play important causal roles in disease initiation and/or progression , 62. In omics profiles would be a great advantage that allows, in theory, the use of all available genomics data associated with the disease. However, this also imposes a huge responsibility of maintaining consistency in the subtype context across the multiple dimensions of analysis to produce meaningful and concordant results.Further, to understand the cause–effect relationship and the mechanistic overview of PDAC progression, we performed interaction network analysis on GMCs on the basis of interactome information. This multidimensional network was analyzed for topological cues to identify the most probable candidates for KRs of the network and, by extension, key disease modulators. These KR molecules represent regulatory bottlenecks and play a causal role in disease initiation and progression. The presence of these regulatory bottlenecks in establishing disease phenotype has been shown in multiple studies relating to cancer , 63–68. multi-omics integration using new experimental and computational approaches is already producing useful functional models and meaningful insights into disease pathophysiology.In this study we were able to isolate converging regulatory modules and KR molecules associated with PDAC pathophysiology by employing a hierarchical systems-level multidimensional data analysis approach. GSEA on subnetworks of these KRs depicted significant enrichment in the leading edges of the CGMC network genes, suggestive of their important roles in PDAC progression. The identified KRs were able to differentiate poor survivors from better survivors, further strengthening our evidence that they are genes of prognostic value and can be used as probable therapeutic targets in treatment of pancreatic cancer. Data integration methods for multidimensional data are still in their infancy, but


Epidural spinal cord stimulation has a long history of application for improving motor control in spinal cord injury. This review focuses on its resurgence following the progress made in understanding the underlying neurophysiological mechanisms and on recent reports of its augmentative effects upon otherwise subfunctional volitional motor control. Early work revealed that the spinal circuitry involved in lower-limb motor control can be accessed by stimulating through electrodes placed epidurally over the posterior aspect of the lumbar spinal cord below a paralyzing injury. Current understanding is that such stimulation activates large-to-medium-diameter sensory fibers within the posterior roots. Those fibers then trans-synaptically activate various spinal reflex circuits and plurisegmentally organized interneuronal networks that control more complex contraction and relaxation patterns involving multiple muscles. The induced change in responsiveness of this spinal motor circuitry to any residual supraspinal input via clinically silent translesional neural connections that have survived the injury may be a likely explanation for rudimentary volitional control enabled by epidural stimulation in otherwise paralyzed muscles. Technological developments that allow dynamic control of stimulation parameters and the potential for activity-dependent beneficial plasticity may further unveil the remarkable capacity of spinal motor processing that remains even after severe spinal cord injuries.The online version of this article (doi:10.1007/s13311-016-0421-y) contains supplementary material, which is available to authorized users. Severe spinal cord injury (SCI) is a catastrophic condition, causing disability of vital body functions below the lesion level. While contemporary clinical standards are successfully applied to deal with emergency and secondary complications, and the understanding of spinal cord biology is continuously growing, SCI still cannot be cured and the prognosis for the recovery of meaningful voluntary motor control and locomotion after a clinically complete lesion is very limited . One appEpidural SCS has a long history of application in various motor disorders , 16. It Research into the use of SCS in SCI continued, where the relationship between stimulation site and the spinal level and severity of the lesion were better defined . StimulaThe interpretation of these earlier results was facilitated by increasing knowledge of the neural structures being electrically stimulated. At therapeutic intensities for the management of pain or spasticity , large-to-medium-diameter sensory fibers within the posterior roots , are stiIn people with incomplete cervical lesions, SCS below the lesion was postulated to act through spinal and brainstem levels , 36. It The finding by Dimitrijevic et al.  that theEach stimulus pulse essentially activates motoneurons through monosynaptic connections, to evoke a posterior root-muscle (PRM) reflex when stimulus strength is adequate , 38. ThiWhen SCS is applied continuously, the simplest response type is the stimulus-time locked monosynaptic PRM reflex evoked at 2 Hz  with no modulation or depression within the series of responses Fig.  34, 45], 4534, 4SCS at 22–50 Hz can generate locomotor-like electromyographic (EMG) activity and induce involuntary leg flexion-extension movements in individuals with chronic, motor complete SCI lying supine . Thus, iA recent study explored the capacity of the functionally isolated human lumbar spinal cord to produce a wide range of coordinated rhythmic multimuscle activation patterns under tonic SCS Fig. . StatistIn summary, the motor output generated by lumbar SCS is the integral of repetitively activated mono-, oligo-, and polysynaptic reflex circuits, as well as of the operation of a more complex, plurisegmentally organized network recruited by the tonic nature of the input , 53. TheVoluntary leg movements are largely controlled through the activation and regulation of spinal interneurons , many ofPostmortem anatomical studies have revealed that residual white matter crossing the lesion is a common finding even in those individuals with an SCI classified as clinically complete, that is, without voluntary movement and sensation below the injury –61. CondDescending volitional activation of spinal inhibitory function in paralyzed individuals was demonstrated taking the form of intentional suppression of spinal reflex responsiveness below the lesion level Fig.  64–66].–66.64–66When encouraged to perform a purposeful movement of the paralyzed limbs, some individuals with discomplete SCI are able to generate task-specific EMG activity. Instructed to flex hip and knee joints or, focally, the ankle joint, approximately 10 % of the individuals with discomplete SCI generated traces of motor unit activity, yet not strong enough or insufficiently coordinated to elicit a visible contraction or movement , 65.In conclusion, there is a high likelihood that clinically silent translesional neural connections can survive even a severe SCI. That these neural pathways could provide a basis for recovery through enhancement of residual functions by future treatment approaches was already predicted in early anatomical and physiological studies , 67.Clinically viewed, many cases of augmented function, including improvement in motor strength and voluntary motor function, were reported in the early applications of SCS in MS and SCI , 28, 69.A similar case involving a patient with a chronic, clinically motor complete, and sensory incomplete SCI is presented in Fig. A recent single-case study considerably increased the interest in lumbar SCS as a neuroaugmentative intervention . There, The long periods of stimulation combined with intensive active training additionally raised the question of whether they could have resulted in improved functional, translesional connections with the lumbar circuitry , 71, as Sensory feedback input from the legs to the lumbosacral spinal cord induced by externally imposed stepping motions on a treadmill can generate step-phase synchronized EMG activity in individuals with SCI who are not able to produce similar motor patterns voluntarily , 55, 73.New therapeutic paradigms for use in neurological disorders have evolved out of engineering advancements coupled with an increasing understanding of the underlying physiology . In receESM 1(PDF 1224 kb)Below is the link to the electronic supplementary material.


Pathogens grow and cause disease by exploiting the host as a rich and diverse source of food. However, it is not always an easy task to tap these food resources since the host innate immune response restricts pathogen access to crucial nutrients  . PathogePathogens utilize various nutrients at vastly different rates. Some nutrients such as metal ions, cofactors, and monomeric components of proteins, lipids, and carbohydrates are directly incorporated into biomass. In addition, pathogens need to degrade substantial amounts of nutrients to small excreted waste products in order to obtain the energy they require for assembling biomass components and maintaining homeostasis (such as counteracting dissipation of membrane gradients). Uptake and metabolism of such energy sources is generally much faster compared to nutrients that are directly incorporated into new biomass.While extracellular pathogens can often exploit rich energy sources delivered to them by the host circulation, intracellular bacterial pathogens depend on their surrounding host cells for supply of energy sources at sufficiently high rates. This extensive metabolic interplay between host cells and the pathogens that they nurture is likely full of fascinating, rich biology. However, these major fluxes remain poorly characterized since common methods to study pathogen metabolism such as tracking incorporation of isotope-labelled carbon/nitrogen into biomass are not informative on nutrients converted into excreted waste products.On the other hand, new approaches start to unravel how intracellular pathogens acquire energy sources at sufficiently high rates for growth and disease—in particular, intravacuolar pathogens that must import nutrients across the vacuolar membrane. This Pearl article will highlight acquisition of energy sources by intravacuolar pathogens and its role in disease. For other aspects of microbial nutrition in vivo and host mechanisms for nutrient restriction, the reader is referred to various recent reviews –5.Escherichia coli, generating a daughter cell requires hydrolysis of some 8 x 109 adenosine triphosphate (ATP) molecules to assemble biomass and support essential maintenance requirements, even if all monomeric components  are freely available [E. coli (about 37% of glucose is used for ATP generation) [Bacterial proliferation requires high amounts of energy. For bacteria such as vailable . Even ineration) ). PathogShigella cells that rapidly grow in the cytosol of a human epithelial cell would completely consume the most abundant host metabolites within just a few minutes [For extracellular pathogens with direct access to blood or interstitial fluid, host glucose and glutamine provide rich energy sources that are rapidly replenished by the host circulation. In contrast, intracellular pathogens access diverse host cell metabolites, but these nutrients are quickly exhausted if not actively replenished by the host cell. As an example, ten  minutes ,7). TherWhat nutrients can be delivered by host cells at sufficiently high rates to meet the energy demands of intracellular pathogens? The host cells mostly depend on abundant blood metabolites, in particular glucose and glutamine, but also lactate in areas with limited oxygenation . Host ceE. coli (EIEC) can directly use incoming glucose  and low transport rate [E. coli, which has at least two high-affinity pyruvate transporters (KM in the range of 10 μM) [It is important to note that in primary cells with sufficient oxygenation, host cell mitochondria also take up pyruvate for fueling the tricarboxylic acid (TCA) cycle. Pathogens will thus compete with the mitochondria for the cytoplasmic pyruvate pool . This imort rate , comparef 10 μM) . PathogeCoxiella resides in phagolysosomes, where it resists the highly adverse conditions and captures amino acids released from proteins as part of the normal host cell protein turnover [Anaplasma phagocytophilum uses the type IV-translocated effector 1 (Ats-1) to promote the host autophagy degradation pathway and gain access to amino acids [Legionella pneumophila also use amino acids (or amino acid-derived pyruvate) as the main intracellular energy sources that are metabolized in their TCA cycle [Legionella injects into the host cell the type IV-translocated Ankyrin B (AnkB) effector, which functions on the pathogen-containing vacuole (PCV) as a platform for the assembly of polyubiquitinated proteins, which are targeted for proteasomal degradation [Legionella growth within the PCV, and this growth defect is totally bypassed upon supplementation of cysteine (Cys), serine (Ser), alanine (Ala), pyruvate, or citrate, all of which feed the TCA cycle [In addition to exploiting host cell nutrient uptake, or host metabolism, some intravacuolar pathogens employ diverse sophisticated mechanisms to exploit valuable nutrients released by host cell degradation of polymeric biomass components, in particular proteins . Coxiellturnover . Anaplasno acids . In contCA cycle . To mobiradation . InhibitCA cycle .Salmonella access many diverse host nutrients in infected mouse spleen [Salmonella obtains carbohydrates such as N-acetylglucosamine [Salmonella suggest that instead of one major energy source, the host–pathogen metabolic interface can be much more complex, with a diversified portfolio of energy sources. Interestingly, glycerol generated from lipid degradation and/or glycolytic intermediates can also be a major energy source for intracellular pathogens . Interestingly, Francisella boosts the levels of free Cys in the host cell cytosol using its γ-glutamyl transpeptidase (Ggt) enzyme to cleave host glutathione (GSH) (L-γ-L-glutamyl-L-Cysteinyl-glycine) [Legionella is auxotrophic for several amino acids , five of which are essential for human cells [There is an emerging theme that many sources of energy, and amino acids in particular, are essential for intracellular pathogens as well as their host cells. Human cells are auxotrophic for nine amino acids , while Cys is semiessential and is the most limiting amino acid in human cells. Therefore, intracellular pathogens have evolved with nutritional strategies to enhance the level of these essential sources of energy. The cytosolic pathogen, glycine) . Similaran cells . We specan cells ,31.Plasmodium and Toxoplasma (see above), evidence for bacterial pathogens suggests participation of host solute-carrier (SLC) transporters, the second largest superfamily (~400 putative transporters) of membrane proteins in humans [+- or H+-coupled symporters, and antiporters, located in cellular and organelle membranes. About 25% of all SLCs are members of seven SLC families that transport amino acids, but other substrates such as glucose, lipids, and drugs are also transported by specific SLCs.Intravacuolar pathogens are faced with the additional challenge of importing nutrients across the vacuolar membrane. However, there is very limited knowledge of how intravacuolar pathogens import nutrients from the host cell cytosol across the vacuolar membrane and into the lumen of the pathogen-containing vacuole . In addin humans . The SLCSalmonella and Mycobacterium within macrophages, where it imports Arg across the pathogen-containing vacuolar membrane [Legionella [Legionella PCV within human macrophages indicated the presence of a few SLCs that transport various amino acids [Toxoplasma pore). Finally, PCVs might extensively communicate with other cellular vesicles, exchanging luminal contents. This has been documented for Salmonella, which acquires extracellular nutrients through stealing cargo of normal host cell endocytosis [In particular, it has been shown that the host cationic amino acid transporter SLC7A1 is acquired by the PCV-harboring membrane . SLC1A5,gionella , but it no acids , and trano acids . In addiocytosis ,10.It is clear that energy supply is one of the most crucial aspects determining pathogen growth and virulence. However, still only a small minority of pathogen metabolism studies are focused on this important issue . The claHowever, separation of host and pathogen metabolites to directly determine participation of host and pathogen pathways remains challenging because of the very short turnover time, which can result in substantially altered metabolomes during pathogen purification attempts. New techniques that can follow metabolites in a spatially resolved manner would offer fascinating opportunities to overcome these difficulties. In the meantime, specific perturbation of host and/or pathogen enzymes can provide important insights on relevant pathways and their localization in the host or pathogen cells. Once major energy sources have been unraveled, host pathways that supply them at sufficiently high rates can be investigated. In many cases, such pipelines are manipulated by the pathogen as a major part of their molecular virulence mechanisms.Our knowledge of metabolic host responses to bacterial pathogens during infection is still limited because of the major experimental challenges of the infection model and the complicated analytical tools and methods. Many studies have utilized metabolic flux analysis revealing host cell metabolic alterations and carbon fluxes during infection by various pathogens  and glutamine (Gln), which are imported by SLC transporters . A two-e


Renal cell carcinoma (RCC) accounts for approximately 3% of all cancer cases. RCCs usually metastasize to the lungs, bones, liver, or brain. Only <1% of patients with bone metastases manifested clavicular RCC metastases. Thus, clavicular metastasis as the initial presentation of RCC is extremely rare. We report a patient with RCC metastasis to the left clavicle, which was first presented with pain caused by a pathological fracture. Magnetic resonance image revealed a renal tumor, and technetium-99m–methylene diphosphonate bone scintigraphy showed multiple osseous metastases. The patient eventually underwent surgery to remove the lateral end of the left clavicle and right kidney. Histopathology revealed renal tumor and clear cell carcinoma in the clavicle. Finally, we review 17 cases of clavicular metastases originating from different malignancies. Bone metastasis of renal cell carcinoma (RCC) is not rare but usually located in the spine, the pelvic bones, or the ribsA 64-year-old man was admitted to the Fourth Hospital of Hebei Medical University in January 2014, due to constant pain in the left shoulder after a mild trauma. X-ray and magnetic resonance imaging (MRI) results revealed a pathological clavicular fracture (The patient underwent surgery to remove the lateral end of the left clavicle. Histopathology of the clavicle revealed clear cell renal carcinoma (--,-,,et al.,,,RCC represents approximately 3% of all cancer cases. Classic symptom triad comprising hematuria, abdominal mass, and flank pain is found in only 10% of patients. However, RCCs commonly metastasize widely before any symptom is observed-,-,-,,,,et al.Clavicular fractures represent 5% to 10% of all fracturesIn conclusion, clavicular symptoms may be manifested before the diagnosis of primary tumors, such as RCC. Clavicular fracture may be the first symptom of tumor metastases to bones. Distinguishing pathological fractures from clavicular fractures due to other causes may help diagnose the primary tumors and necessitate whole-body scanning at an early stage.