With a special focus on systems that have been tested in vivo, this review is an essential summary of the most advanced developments in this rapidly evolving field.Current cancer therapies, including chemotherapy and radiotherapy, are imprecise, non-specific, and are often administered at high dosages - resulting in side effects that severely impact the patient's overall well-being. A variety of multifunctional, cancer-targeted nanotheranostic systems that integrate therapy, imaging, and tumor targeting functionalities in a single platform have been developed to overcome the shortcomings of traditional drugs. Among the imaging modalities used, magnetic resonance imaging (MRI) provides high resolution imaging of structures deep within the body and, in combination with other imaging modalities, provides complementary diagnostic information for more accurate identification of tumor characteristics and precise guidance of anti-cancer therapy. This review article presents a comprehensive assessment of nanotheranostic systems that combine MRI-based imaging  with therapy , connecting a range of topics including hybrid treatment options ( Cancer is a leading cause of mortality, with 606,520 cancer-related deaths projected to occur in the United States in 2020 Figure 1TM and AbraxaneTMTargeting of nanoparticles to tumor sites can be achieved by several general methods: passive, active, or through externally applied forces, as shown in vβ3, a cell adhesion receptor that is overexpressed in several types of cancer e.g. folic acid) e.g. DNA or RNA) and tumor surface receptors, with high specificity and affinity Active targeting utilizes specific interactions between a ligand on the nanoparticle surface and a biomarker, or receptor, on the target cells The use of an external force, such as in magnetic targeting of magnetically-responsive carriers, however, can yield results that are independent of the complex molecular tumor biology e.g. optimized therapeutic windows, dosages, etc.) by using imaging tools to monitor cancer heterogeneity and adaptability in vivo and obtain structural, functional, and molecular information about the tumor region include magnetic resonance imaging (MRI), computed tomography (CT), fluorescence imaging and positron emission tomography (PET), which differ in detection sensitivity, penetration depths, and image resolution 29.Theranostic nanoparticles, having integrated therapy and diagnostics in a single platform, have been developed that could potentially provide a more personalized treatment , MRI-based diagnostics , and tumor targeting approaches (active and magnetic targeting), as illustrated in Figure 2, with a focus on in vivo studies.In this review paper, we discuss recent developments of “all-in-one” nanotheranostic systems that integrate therapy , intercalating within DNA and stopping its replication (e.g. Doxorubicin), or forming intra- and inter-strand DNA crosslinks and restricting DNA repair (e.g. Cisplatin) e.g. cardiotoxicity) e.g. blood-brain-barrier in glioma) required for deep tumor penetration; (b) overcoming poor active targeting through dual receptor targeting or use of multiple tumor-targeting strategies; (c) enhancing antitumor efficacy through dual-drug delivery; and (d) overcoming limitations of MRI through multimodal imaging. A summary of the most important outcomes is shown in Table 1.The era of chemotherapy, Overcoming barriers: Several MRI-traceable, tumor-targeted delivery systems have been developed to overcome the complex drug delivery barriers present in different types of cancer and provide real-time assessment of therapeutic responses. For example, the disorganized vasculature and enriched tumor stroma of pancreatic cancers can potentially be overcome by targeting the urokinase plasminogen activator receptor (uPAR) et al. developed uPAR-targeted SPIONs for the delivery of the drug Gemcitabine to uPAR-overexpressing pancreatic tumor and stromal cells et al. explored the potential of Convection Enhanced Delivery (CED), a minimally invasive surgical strategy that generates a pressure gradient at the tip of an infusion catheter placed directly into the brain tumor (i.e. bypasses the BBB), to deliver high concentrations of therapeutic agents to focal areas of the brain while reducing systemic toxicity The blood-brain-barrier (BBB), which protects the brain from harmful pathogens, represents another major biological barrier for anticancer treatment. Various methods to bypass or increase permeability across the BBB have been summarized by Israel and colleagues e.g. pH- and enzyme) stimuli-responsive, exogenous (e.g. light and magnetic field) stimuli-responsive and multi-stimuli responsive nanomaterials 7K(R2)2 was conjugated to the surface of PTX/SPION-loaded liposomes and the pH-responsive, tumor-targeting, and anti-tumor activity properties validated using in vitro assays and in vivo MRI studies 3; 90% tumor growth inhibition) at day 31 after implantation compared to mice treated with non-targeted nanoparticles . A more thorough discussion on stimuli-responsive cancer theranostic nanocarriers can be found in a recent review by Mi Stimuli-responsive nanomaterials that bypass the biological barriers present in the tumor microenvironment have also been developed for targeted intracellular drug delivery. These nanomaterials can be divided into three categories: endogenous  passive targeting via the EPR effect, 2) active targeting of αvβ3 integrin through RGD grafting, 3) magnetic targeting, and 4) dual active- and magnetic-targeting. The combination of active and magnetic targeting resulted in the highest tumor accumulation (>2.5-fold compared to other groups), the greatest contrast in T2 MRI images, and most potent anti-cancer activity with median survival time of 21.5 days . Similar results were observed by Yang et al., who developed multilayered magneto-vesicles (MuMVs) modified with RGD and DOX and evaluated its efficacy, with and without magnetic targeting, in U87MG tumor-bearing mice vβ3-integrin positive HeLa tumors Dual active- and magnetic-targeting strategies have also been explored to improve MRI detection and enhance anti-cancer efficacy compared to active or magnetic targeting alone Li and co-workers went one step further by combining magnetic and active targeting with a nuclear targeting mechanism et al. developed a theranostic self-targeting nanoplatform by coating DOX-conjugated IONPs with various types of cracked cancer cell membranes (CCCM) derived from homologous tumors Functionalizing nanoparticles with cell-derived membranes as a tumor self-targeting strategy has been explored as an alternative means of targeting by reducing nonspecific uptake by the immune system and enhancing tumor delivery Improving chemotherapeutic efficacy: Combination chemotherapy, with two or more anti-cancer agents, has been shown to enhance therapeutic efficacy compared to monotherapy et al. used Ferumoxytol with a prostate-specific membrane antigen (PSMA)-targeting peptide and two drugs , co-retained within its polymeric coating, for targeted delivery to castration-resistant prostate cancer et al. developed dual active- and magnetically-targeted magnetic PLGA nanoparticles carrying both PTX and curcumin drugs e.g. P-glycoproteins) responsible for cancer drug resistance et al. developed stimuli-responsive superparamagnetic iron oxide nanorods (SIONR) encapsulating pluronic F127, a stimuli-responsive block copolymer that inhibits P-glycoproteins and controls the release of PTX in the tumor area, to improve colon cancer therapy Another strategy to improve the clinical efficacy of chemotherapy is by targeting transporter-based efflux pumps , which can be difficult to distinguish from other artifact signals caused by pathogenic conditions such as internal bleeding, calcification, metal-deposits or air-tissue boundaries i.e. bright) contrast agents. Liu et al. developed poly(lactic acid)-based particles with conjugated drug sorafenib, that were targeted to vascular endothelial growth factor receptor-overexpressing cancer and contained gadolinium (Gd) ion chelates for T1 MRI contrast ®. Additionally, the in vivo antitumor effect of the nanomaterial in hepatocellular carcinoma (H22) tumor was markedly higher compared to free sorafenib solution.In vivo anti-tumor efficacy studies demonstrated significantly enhanced median survival time for C26 tumor-bearing mice treated with Apt-NPs (42 days) versus mice treated with sucrose 10% (21 days), free DOX solution (24 days), and NPs (34 days). In addition, T1-weighted MR images also showed higher accumulation of Apt-NPs in the tumor site in comparison to NPs without aptamers et al.71Recent concerns of nephrotoxicity and long-term brain deposition in patients exposed to Gd has encouraged researchers to develop safer alternatives to Gd In vivo NIRF and T2 MRI images demonstrated the enhanced uptake of MTX-PEG-CS-IONPs-Cy5.5 in folate-overexpressing HeLa tumors compared to PEG-CS-IONPs-Cy5.5, while providing improved anti-cancer treatment efficiency with reduced side effects et al. developed theranostic multimodal liposomes integrated with SPIONs, quantum dots, and the therapeutic agent cilengitide to target and inhibit overexpressed integrin receptors on C6 glioma cells under magnetic targeting In vivo magnetic targeting resulted in enhanced tumor delivery of the agent and effective inhibition of tumor growth, while dual MRI-NIRF imaging allowed for accurate resection of glioma tumors.While MRI provides excellent soft-tissue contrast and high image resolution, it suffers from low sensitivity in differentiating healthy and malignant tissues 3O4 conjugated to the anticancer drug bovine lactoferrin and modified with locked nucleic acid-modified aptamers to provide simultaneous targeting to epithelial cell adhesion molecule (EpCAM) and nucleolin. Oral administration of the targeted formulation improved the survival rate in triple-positive  colon cancer xenograft model, with only 10% of mice experiencing tumor recurrence compared to 30% of mice orally fed with non-targeted nanoparticles In another study, Roy and co-workers developed NIRF/MRI/CT trimodal Feet al. developed magnetic gold nanoparticles (MGs) conjugated with tumor-targeting peptide, LyP-1 (CGNKRTRGC) (MGFs-LyP-1) for targeted trimodality imaging and autophagy-mediated chemosensitization of HepG2 tumor-bearing nude mice Nanoformulations that provide dual T1- and T2-weighted MRI contrast agents have also been developed to enhance MRI sensitivity et al.Gene therapy, which involves the delivery of nucleic acids to modulate the expression of genes involved in tumorigenesis, is a promising cancer treatment modality, especially in conjugation with radiotherapy or chemotherapy In vivo MRI studies demonstrated significant accumulation of siPLK1-carrying SPIONs in the tumor site and efficient PLK1 silencing that significantly inhibited tumor growth. MRI visibility of the formulation allowed for high-resolution spatiotemporal tracking and precise guidance for gene delivery and assessment of treatment efficacy.Gene therapy can be accomplished by upregulating tumor suppressor genes or gene knockdown using siRNA e.g. surviving and MDR1), and oncolytic virotherapy et al. synthesized sphere-like (S-M-MSNs) and rod-like magnetic mesoporous silica nanoparticles (R-M-MSNs) loaded with herpes simplex virus thymidine kinase/ganciclovir and compared their performance in MRI-guided, magnetically-targeted and hyperthermia-enhanced suicide gene therapy of hepatocellular carcinoma tumor-bearing mice Other therapeutic strategies employed include suicide gene therapy, anti-angiogenic gene therapy, genetic immunopotentiation, silencing multi-drug resistance-associated genes  conjugated with 6-(2 nitroimidazole) hexylamine and loaded with magnetized adeno-associated virus serotype 2 (AAV2) and lactate oxidase (LOX) for controlled release of magnetized AAV2 in the hypoxic, lactate-rich tumor microenvironment in vivo through light-triggered virotherapy. A summary of the discussed probes used in gene therapy is shown in Table 2.In oncolytic virotherapy, viruses are modified to selectively grow and replicate in highly susceptible cancer cells, altering the cancer cell's transcriptional and translational machinery, while leaving normal cells unharmed th century, when doctors utilized fever therapies (e.g. through the administration of living bacteria to cancer patients) or circulating heated water to shrink tumors Figure 3, have been used in thermal therapy and we will examine the use of MRI-guided, tumor-targeted nanoparticles in (i) Photothermal therapy (PTT); (ii) Magnetic hyperthermia treatment (MHT); and (iii) ultrasound (US)- and radiofrequency-induced thermal therapies.Thermal therapy, which involves heating tumors, represents another choice for cancer treatment. The benefits of thermal therapy were first realized in the 19Photothermal therapy (PTT): PTT involves the absorption of electromagnetic radiation, typically from a NIR laser, to locally produce thermal energy that induces cancer cell necrosis or apoptosis i.e. dosage of light delivered) and the concentration and photothermal conversion efficiency of the photothermal agent et al.et al.et al. developed a targeted, nanotheranostic agent for synergistic PTT and MRI/fluorescence imaging of cancer by conjugating both HA and CuInS2-ZnS quantum dots on the surface of Fe3O4@prussian blue core-shell nanoparticles in vivo PTT efficacy (tumor growth inhibition >90%) against HeLa tumors compared to controls.Photothermal agents based on organic, noble metal, carbon, or semiconductor materials have all shown good conversion efficiency in the NIR range et al. developed an upconversion luminescence/MRI-guided photothermal agent consisting of upconversion nanoparticles as the core, a layer of SPIONs as the intermediate shell, and a layer of gold as the outer shell in vivo breast cancer model resulted in 100% tumor elimination. Magnetic nanomaterials have also been combined with small photothermal agents, such as indocyanine green (ICG) - an FDA approved dye used in fluorescent-guided surgery et al. developed core-shell magnetite nanoclusters with a coating of polydopamine, and further conjugated PEG and ICG to the surface Because iron oxide nanoparticles tend to have a low molar absorption coefficient in the NIR range, they are often combined with other photoactive nanomaterials or small molecules for effective PTT et al. used MRI-tracing to numerically model the temperature distribution in the tumor region as a pre-treatment tool e.g. Ag2S quantum dots and single-walled carbon nanotubes) with peak absorbance in the second NIR (NIR-II) biological window (1000-1350 nm) for enhanced tissue penetration et al. generated core-shell nanoparticles consisting of a double layer of Au/Ag alloy on the surface of IONPs, which exhibited broad NIR absorption that extended to the NIR-II window with a photothermal conversion efficiency of ~28.3% at 1064 nm -2 1064 nm diode laser. A digital NIR thermal camera also showed significantly increased temperatures of 45.4 °C in the group treated with the magnetic field versus the group without magnetic targeting (38.3 °C). Photoacoustic imaging (PAI), which detects the ultrasound waves generated by thermoelastic expansion of photothermal agents after absorption of NIR energy, has also been used to monitor temperatures induced by PTT. Several probes have been developed that combine three or more imaging modalities , including ICG-modified iron nanoparticles for MRI/PAI/fluorescence-guided PTT of MCF-7 xenografted breast tumors 111. An extensive review of combinations of PTT and PAI is provided by Ge and colleagues Various approaches have been explored to further enhance PTT efficacy. Beik et al. developed a probe consisting of (i) transferrin for active targeting and entry into cancer cells through receptor-mediated endocytosis, (ii) TAT peptide, which binds the importing α and β receptors for nuclear entry, and (iii) fluorescent dyes (fluorescein isothiocyanate or cyanine7) linked to IONPs for MRI/fluorescence-guided PTT Another strategy to enhance PTT efficacy is through targeting to the cell nucleus. The nucleus, which governs metabolism, reproduction, and the cell cycle, is considered the ultimate and most sensitive organelle for subcellular PTT targeting 2O2 to highly toxic reactive oxygen species . Iron ions are known to catalyze the formation of ROS et al. showed that IONPs with mitochondria-targeting iridium(III) complexes (Ir@Fe3O4 NPs) rapidly damaged cancer cells under photoactivation 3O4 NPs at the tumor site and NIR laser irradiation resulted in a mild localized temperature increase to 42 °C and the production of •OH radicals, which efficiently killed cancer cells both in vitro and in vivo.While standard PTT attempts to achieve a tissue temperature >50 °C, which can also damage normal surrounding tissues Magnetic hyperthermia treatment (MHT): MHT utilizes heat generated by MNPs under an alternating magnetic field to selectively kill tumor cells et al. estimated that for particles with a specific absorption rate (SAR) of 1,000 W/g and for a 5 mm diameter tumor, a minimum concentration of 650 µg/cm3 is required to achieve sufficient hyperthermia et al. developed Mn-Zn ferrite magnetic nanocrystals modified with ICG dye and RGD peptide as a tumor-targeted, multimodal (T2*-weighted MRI/fluorescence imaging) contrast agent for MHT Ultrasound (US) and radiofrequency (RF)-induced thermal therapy: Due to the limited tissue-penetrating depth of light, PTT is restricted primarily to the treatment of superficial tumors et al. fabricated temperature-responsive, anethole dithiolethione-doped magnetic nanoliposomes (AML) loaded with SPIONs and a hydrogen sulfide (H2S) pro-drug that transformed into micro-sized H2S microbubbles in the tumor tissue upon US irradiation 2S bubbles, in addition to significantly enhanced anticancer efficacy in vivo.et al. developed anti-EGFR monoclonal antibody-conjugated SPIONs as a novel approach to improve the MRI sensitivity and ultrasonic energy deposition at low US power MRI-guided focused US (MRgFUS) is a clinically approved procedure that uses anatomical information from MRI scans to localize, control, and target US energy to ablate the target tissue 3O4 nanoparticles, fullerene (C60), and DTX was developed for RF-triggered drug release and combined chemotherapy-thermal therapy Table 3.Radiofrequency thermal therapy (RTT) is another form of anticancer thermal therapy that relies on the conversion of radiofrequency (RF) energy into heat in the presence of RF absorbing agents. A folate-targeted, thermosensitive liposomal system incorporating FePhotodynamic therapy (PDT): PDT is a laser-based therapy that utilizes light to excite photosensitizers, which transfer the energy to neighboring oxygen molecules and generates highly toxic ROS et al. developed NIR-light activated upconversion nanoparticles conjugated with chlorine6 and RGD peptide to target αvβ3-integrin overexpressing human U87 glioma tumor et al. developed dextran nanoparticles loaded with Fe3O4 nanoparticles for NIRF/MRI dual imaging and magnetic targeting, and conjugated with redox-responsive chlorin e6 (Ce6) for PDT treatment of human breast carcinoma (MCF-7) 3+ for PDT therapy of pancreatic tumor xenografts In vivo fluorescence imaging and T1-weighted MRI demonstrated significant accumulation of Ce6 and Gd3+ at the tumor site and resulted in enhanced PDT efficiency.Sonodynamic therapy (SDT): SDT is similar to PDT but it utilizes US to activate specialized agents known as sonosensitizers to generate ROS to kill cancer cells et al. integrated ferrate(VI) and protoporphyrin IX into organosilica nanoparticles for sensitizing SDT of hypoxic tumors 2 nanoparticles to alleviate hypoxia by converting tumor-overexpressed hydrogen peroxide into oxygen radicals et al. developed a tumor-targeted nanosonosensitizer by integrating manganese oxide in hollow mesoporous organosilica nanoparticles loaded with the organic sonosensitizer (protoporphyrin) and decorated with the RGD peptide 2+ under the reducing environment, and an efficient 96% tumor inhibition rate in mice after SDT treatment was observed.Ferroptosis: Ferroptosis is a newly discovered form of cell death caused by the accumulation of large amounts of iron in the cell and characterized by the formation of lipid ROS, ultimately causing cell death 3O4/Gd2O3 hybrid nanoparticles were used to accelerate cancer-cell death in orthotopic brain tumors through a ferroptosis-like mechanism vβ3-mediated endocytosis, which resulted in significant inhibition of tumor growth, monitored by MRI. In another study, platelet membrane-camouflaged Fe3O4 nanoparticles were used for combination ferroptosis and immunotherapy to combat tumor metastasis Table 4The heterogeneous and multifactorial nature of cancer requires a multimodal therapeutic approach to overcome drug resistance, reduce the necessary dose, and minimize side effects Chemotherapy in combination with thermal therapies: Despite its minimal invasiveness, improved precision, and reduced side effects compared to chemotherapy et al. developed Ag-Fe3O4-based nanoparticles modified with folate for dual MRI/NIRF-guided chemotherapy-PTT that demonstrated an anti-tumor effect greater than that obtained with monotherapies et al. developed a magnetically-targeted theranostic nanosystem loaded with DOX for combined MHT and magneto-thermally-responsive drug release to minimize the toxic effects and enhance the targeting efficacy of chemotherapy for liver cancer treatment MHT is most effective when used as an adjunct to chemotherapy and radiotherapy, where increased tumor perfusion by the elevated temperatures improves chemotherapeutic delivery and increases oxygen concentrations for enhanced radiotherapy (tumor radioresistance is primarily due to hypoxia) et al. developed chitosan-based αvβ3-targeted nanomicellar agents comprised of DOX-SPION complexes for combined chemotherapy and MHT against orthotopic TNBC in a mouse tumor model Metastatic triple-negative breast cancer (TNBC), lacking estrogen, progesterone, and Her-2 receptors, is the most aggressive form of metastatic breast cancer and with therapy options limited to chemotherapy, the overall survival remains poor at 13-18 months Targeted chemotherapy has also been used following high-intensity focused ultrasound (HIFU) ablation, which often results in incomplete eradication of deep-seated tumors, and has common side-effects including damage to nearby healthy tissue and skin burn Chemotherapy in combination with PDT: Feng et al. developed magnetic manganese oxide nanospheres for magnetically-guided combined chemotherapy-PDT et al. utilized PTX to induce self-assembly of human serum albumin nanoparticles modified with RGD (targeting), Ce6 photosensitizer (PDT and fluorescence tracking), and Mn2+ for MRI tracking vβ3-integrin-positive U87MG tumors compared to the monotherapies. In another example, Feng et al. developed DOX-loaded hollow mesoporous copper sulfide (CuS) nanoparticles capped with SPIONs for magnetically targeted, combined PTT-PDT-chemotherapy e.g. carboxylic acids and tertiary amines), acid-labile chemical bonds , anionic and cationic pH-responsive polymers, and pH-sensitive peptides (e.g. GALA peptide) et al. developed DOX-encapsulated PLGA, with Ce6 (PDT and fluorescence imaging), Gd-DTPA (T1 MRI tracking) and folate (targeting), as a tumor-targeted, charge-switchable nanomaterial with multistage pH-sensitive behavior for dual chemotherapy-PDT of cancer  et al. developed black phosphorus quantum dots gated, carbon‐coated Fe3O4 nanocapsules with pH, NIR, and redox triple-stimuli responsive behavior for continuous DOX release at the tumor site The variation of pH in the tumor microenvironment (pH 6-7), endosomes (pH 5-6), and lysosomes (pH 4-5) has also been exploited for the delivery of chemotherapy to tumors Combination of PTT and PDT: Cheng et al. developed ultrasmall Gd2O3 nanoparticles that were labeled with HA (for active tumor targeting), coated with polypyrrole , and conjugated with aluminum phthalocyanine chloride for combined PTT-PDT and fluorescence et al. encapsulated ICG, a dual photothermal agent and photosensitizer, in a TiO2:Yb-based nanoparticle for trimodal luminescence/MRI/fluorescence imaging and combined PTT-PDT et al. developed a nucleus-targeting nanoplatform for dual PTT-PDT that resulted in high DNA damage e.g. triphenylphosphonium and cyclometalated Ir(III) complexes), peptides and aptamers , a polydopamine inner shell (for PTT), and ICG (combined PDT and PTT) encapsulated in mesoporous silica. Additional surface modification with transferrin and triphenylphosphonium, a lipophilic cation, enhanced tumor accumulation  and further targeting to mitochondria resulted in significantly higher cytotoxicity against an orthotopic lung cancer model.Targeting subcellular organelles, including the nucleus, mitochondria, and lysosomes, has been shown to significantly enhance phototherapeutic efficacy Chemotherapy in combination with gene therapy: Chemotherapy is often hindered by the immunosuppressive tumor microenvironment that impedes drug delivery and results in drug resistance et al. developed a nanotheranostic system carrying siTGF‐β that down-regulated TGF-β gene expression in glioblastoma and sensitized the tumor to temozolomide-based chemotherapy et al. developed a tumor cell-derived extracellular vesicle (TEV)-based nanosystem, containing gold-iron oxide nanoparticles, for targeted delivery of anti-miRNA-21 to treat 4T1 breast tumors in mice As tumorigenesis is associated with abnormal microRNA (miRNA) expression Combination of immunotherapy with other therapies: Immunotherapy leverages a patient's immune system to destroy malignant cells et al. developed an anti-PD-L1 antibody-conjugated liposomal cerasome (i.e. ceramic-coated liposome) loaded with PTX for combined immuno-chemotherapy T1 and CT26 tumors. In addition, the targeted nanoparticles demonstrated significantly enhanced antitumor activity compared to nontargeted nanoparticles, free PTX, and free PD-L1 antibody. The results suggested that immunotherapy functioned as an adjuvant to improve the efficacy of chemotherapy. In another study, a nanoplatform consisting of SPIONs, anti-PD-1 antibody, perfluoropentane, and GRGDS peptides showed effective ablation of melanoma with combination immunotherapy and PTT et al. designed iron oxide nanorings for combined immunotherapy and MHT to eradicate primary tumors and inhibit metastatic spread e.g. dual magnetic-active targeting), combination therapies (e.g. combined photo-chemotherapy), and unique imaging opportunities .Much of the effort to improve the quality of patient care has focused on improving the “targeting” of cancer therapies, by exploiting the molecular signature of cancer cells, to deliver the optimum drug dosage to tumors without harming healthy cells. Nanotechnology offers the opportunity to combine drug targeting with biomedical imaging, specifically MRI with its high spatial resolution, and other treatment modalities to overcome the challenges of cancer diagnosis and therapy. This review paper describes recent developments of multifunctional, cancer-targeted nanotheranostics comprised of targeting molecules, imaging, and therapeutic agents for MRI-based diagnosis and treatment of in vivo tumors. A variety of interdisciplinary topics are covered ranging from novel targeting strategies  offer some level of targeting and dose control, they also bring an extra level of complexity that complicates clinical translation and some are limited in their depth of penetration. Proper treatment planning must, therefore, not only consider the strengths and limitations of each modality, but also the physiology, distribution, and type of cancer to be treated.Other challenges for clinical translation of novel contrast agents is the high dose necessary to achieve the desired diagnostic and therapeutic response, which creates safety and toxicity issues. In addition, the low utilization rate and poor market performance of prior FDA-approved, nanoparticle-based MRI contrast agents, including ferucarbotran and ferumoxides due to the availability of better alternatives et al. showed that actively targeted sub-5 nm IONPs experienced a 6-fold increase in accumulation compared to non-targeted sub-5 nm IONPs, while active targeting of 30-nm IONPs showed a modest 1.15-fold increase in accumulation compared to non-targeted 30 nm e.g. nanoparticle's physiochemical properties and architecture, ligand type, and density) in vitro, which may not be representative of what is present in a patient's tumor. A better strategy would be to correlate biomarker expression to tumor grade of human samples. Magnetic targeting is another option, but very few clinical trials have been conducted - mainly due to lack of effective mechanisms for precise delivery of magnetic gradients required to navigate nanoparticles against blood flow (hydrodynamic drag forces in large vessels is significantly greater than magnetic forces) and to deep-seated tumors in vivo models.The majority of targeting strategies discussed in this paper rely on active targeting to “overexpressed” tumor receptors, which requires that the nanoparticles first reach the desired target, mainly by escaping the vasculature through leaky blood vessels, to take advantage of the increased affinity between the carrier and target. In fact, it is generally accepted that, compared to active targeting which can provide a high signal-to-noise ratio for biomarker detection, the permeation of particles through leaky vasculature plays a more dominant role in overall NP accumulation


Despite decades of intensive basic and clinical research efforts, there is still no successful vaccine candidate against human immunodeficiency virus (HIV-1). Standard combined antiretroviral therapy (cART) has been successfully developed and has given remarkable results suppressing HIV-1 infection and transmission. However, cART cannot fully clear the virus from the infected patients. A cure for HIV-1 is highly desirable to stop both the spread of the virus in humans and disease progression in HIV-1 patients. A safe and effective cure strategy for HIV-1 infection will require appropriate animal models that properly mimic HIV-1 infection and advance HIV-1 cure research. Animal models have been a crucial tool in the drug discovery process for investigation of HIV-1 disease mainly in preclinical evaluations of antiretroviral drugs and vaccines. An ideal animal model should recapitulate the main aspects of human-specific HIV-1 infection and pathogenesis with their associated immune responses, while permitting invasive antiretroviral studies. The best humanized mouse models would allow a thorough evaluation of antiretroviral strategies that are aimed towards reducing the establishment and size of the HIV-1 reservoirs. In this review, we evaluate multiple humanized mouse models while presenting their strengths and limitations for HIV-1 research. These humanized mouse models have been tailored in recent decades and heavily employed to address specific quintessential and remaining questions of HIV-1 persistence, pathogenesis and ultimately, eradication. Because of logistical, administrative, and ethical restrictions when working with human donor tissue samples, development of new antiretroviral therapies requires suitable small animal models that accurately mimic the natural course of HIV-1 infection in humans. In general, there is also a limited availability of human tissues  at various stages of the disease, which have been treated with different regimens, and untreated samples. Despite significant contributions of non-human primate models to HIV-1 therapeutic design and vaccine research, these animal models are not fully suitable for in vivo HIV-1 research due to ethical problems and high maintenance costs. Moreover, SIV infection is an inexact model for HIV-1 infection [in vivo. Therefore, immunodeficient mice engrafted with vital human cells and tissues, i.e., “humanized mice”, have become an increasingly popular and utilized option in a variety of HIV-1 preclinical studies. Different technical steps have been used to generate versatile humanized mice, including injection routes, mouse age, irradiation sources, and immunodeficient mouse strains. In this review, we describe three of the most used mouse models for HIV research.Different humanized mouse models have been introduced to enable HIV-1 research Foxnlnu), which results in abnormal fur growth and an athymic phenotype due to defects in thymic stromal development [+T and CD8+T- cells, and therefore were not useful for HIV-1 research but were useful for xenotransplantation studies.Nude mice, first developed in the mid-1960s , carry aelopment . These m+T-cell loss [The first two humanized mouse chimeric hu-SCID models were designed by engrafting the SCID mice 1) with adult human PBLs  and 2) well loss , with a ell loss . The nexell loss . This inell loss .scid mutation into a mouse of non-obese diabetic (NOD) background resulted in the creation of NOD-scid mice missing T-, B-, and NK- cells, which allowed a slightly more complete level of human cell reconstitution [IL2rα) gene into NOD-scid mice, creating NOD-scid-γcnull mice (NSG or NOG mice). These mice exhibited defective responses to mouse cytokines IL-2, IL-4, IL-7, IL-9, and IL-15 [RAG1null and RAG2null) caused more efficient immunodeficiencies including an absence of T-, B-, and NK- cells and moderately impaired dendritic cell (DC) and macrophage cell subsets [in vivo studies . The model is also attractive for viral kinetic studies in vivo due to the strain-dependent differences in kinetics of HIV-1 replication in these mice. This model has been heavily utilized for short-term HIV suppression studies (3 to 4 weeks), but has serious issues, including the development of lethal xenogeneic graft-versus-host disease (GvHD) syndrome and a requirement for a more compromised mouse strain. An upgraded model showed higher human cell engraftment levels [The overall efforts using humanized mice include either gene deletions or backcrosses of strains with mutations that affect T-cells, B-cells, natural killer (NK) cells, macrophages, TLRs, cytokines, and transcription factors. These mutations are meant to minimize the presence of murine cells and enhance the presence of human donor cells and tissues to better mimic human immune responses –16. The titution –19. Greand IL-15 –22. Knoc subsets ,24. Humat levels ,26, but t levels ,28.+ hematopoietic stem cell (HSC) engrafted adult irradiated NSG/NOG mice, then later of the newborn RG, NSG or NOG mice. The newborn version was very attractive due to a much longer human cell lifespan, the superior lasting vitality of originally engrafted human cells [+ engrafted newborn humanized NSG mice mimic critical features of human HIV-1 infection due to generated primary immune responses and the development of a lymphoid-like system of human origin with T- and B- cells, monocytes, plasmacytoid and conventional DCs, lymph nodes, and thymic nodes. The lymphoid tissue contains key latent reservoirs established by HIV-1 during acute infection, opening new options in HIV-1 latency research, including overcoming issues when studying Gut-Associated Lymphoid Tissue (GALT) and Central Nervous System (CNS) infections. The model appears suitable to explore viral distribution in tissues, to sort cell populations to better define virus-cell interactions, and to test new antiretroviral and adjunctive therapies [et al., [The requirement for longer-term studies involving HIV-1 persistence and latency resulted in the creation of first the CD34an cells –31, and an cells . HIV-1 iherapies ,34 in lo[et al.,  showed t+ adult mice undergo productive infection and manifest several aspects of T-cell pathogenesis, indicating a good utility of this model for various new therapeutic interventions [in vivo studies including optimized antiretroviral therapies. NSG mice transplanted with CD34+ cells are useful for the replication of HIV-1 induced pathology and testing different antiviral therapies. This model provides a much more accessible experimental system for studies of viral infection than do models that depend upon transplanting human liver, thymic tissues, and autologous CD34+ HSC (BLT mice). And, as noted above and detailed in We and others have previously shown that humanized NSG- CD34ventions ,30,36–38scid mice; a more recent BLT model uses upgraded mouse strains  [ex vivo (once viremia is reestablished after stopping cART). Importantly, unlike the CD34+T-NSG model, due to robust development of human mucosal immunity (including Peyer’s patches and GALT), the BLT mice are infectable by HIV-1 via vaginal, rectal, and oral routes [+T-cell immune responses (which closely mimic human immune responses), leading to rapid viral evolution, predominantly in CD8+T-cell epitopes [in vivo replication spectrum of viruses isolated from untreated HIV+ patients with undetectable viral loads , showing that these viruses are replication competent and capable of infecting and depleting CD4+T-cells in vivo [+T-cell decline).The bone-marrow-thymus tissues -subrenal capsule transplantation (BLT) model is the most robust and effective current model for HIV-1 studies because these mice have excellent T-cell reconstitution and closely recapitulate a primary immune response against productive HIV-1 infection , 42,43. RG mice) ,45. TheyRG mice) ,47. The RG mice) . Furtherl routes ,49. Thisl routes   can occur. There is an existing difficulty in generating human humoral immune responses leading to class switching and immunoglobulin G (IgG) antibody production. The adult NSG model has a functional limitation. Its lack of multi-lineage human hematopoiesis limits HIV-1 studies to a few weeks’ duration, but that issue has been partially overcome with newer hu-mouse models.+ and CD4+T-cell numbers). Neonatal NSG mice have impaired development of the germinal centers and lymph nodes, important tissues when studying HIV-1 persistence and latency [+T-NSG mice, the antibody responses are quite weak and largely restricted to immunoglobulin M (IgM) due to inefficient class switching, specifically after virus infection [In the neonatal NSG model, some animals may develop more B- than T-cells, rendering those mice useless for HIV-1 studies. Additional issues include the possibility of lacking available mice for control experiments due to timing that includes the several months required for the engraftment, followed by the engraftment evaluation .Further improvements of existing hu-mouse models and more accurate mimicking of the full human immune system are still needed. To improve the earlier listed limitations of the existing hu-mouse models, there are ongoing efforts towards generating new strains of immunodeficient mice transgenic for human HLA molecules. The rationale is that this will strengthen immune responses and the expression of human cytokines and growth factors and thereby improve human cell reconstitution and homeostatic maintenance. There is a need for the inclusion of other human genes responsible for cell differentiation and intracellular interactions. Expression of human growth factors and HLA proteins may help to derive a composite recipient mouse strain having all the desirable features needed for HIV research.Numerous investigators worldwide continue searching for molecular mechanisms to resolve remaining defects in humanized mice, aiming for superior engraftment to overcome the existing deficiencies described in this review. This includes enhancement of human adaptive and innate immunity, and reduction of mouse innate immunity. Mouse models are faster and cheaper than non-human primate models and avoid concerns about reciprocal Fc-FcR evolution, i.e., how effectively human antibodies can drive Fc-mediated effector function in non-human primates. Given the notable progress made using humanized mice for HIV-1 research in recent years, the development of more advanced humanized mouse models in the HIV-1 field, especially for HIV-1 vaccine studies, is of great importance.


Rational: Many efforts have been made to develop ligand-directed nanotheranostics in cancer management which could afford both therapeutic and diagnostic functions as well as tumor-tailored targeting. Theranostic nanoplatform targeting transferrin receptor (TfR) is an effective system for favorable delivery of diagnostic and therapeutic agents to malignancy site.Methods: To enable amalgamation of therapy and diagnosis to many TfR+ tumor, hTfR (human TfR) monoclonal antibody (mAb)-functionalized HPPS nanoparticle (HPPS-mAb) was prepared with hTfR mAb on the shell and with fluorophore DiR-BOA in the core. The targeting specificity was investigated in vitro by immunostaining and in vivo using a double-tumor-engrafted mouse model. HPPS-mAb/siRNA effect on HepG2 cells was determined by RT-PCR and western blot.Results: HPPS-mAb could specifically target cancer cells through TfR and achieve tumor accumulation at an early valuable time node, thus efficiently delivering therapeutic survivin siRNA into TfR+ HepG2 cells and mediating cell apoptosis. DiR-BOA can act as an imaging tool to diagnose cancer.Conclusions: Our studies provide a promising TfR mAb-directed theranostic nanoplatform candidate in tumor molecular imaging and in TfR targeted tumor therapy. An optimized cancer treatment strategy would deliver the right type of therapeutic drugs to the right target, to achieve localized control of the disease efficiently with minimal systemic toxicity. Emerging cancer nanotheranostics offers promising nanoplatforms with diversified capabilities for drug loading and release, for tumor targeting and imaging, and eventually for monitoring cancer therapy non-invasively and in real time. A traditional nanotheranostic agent is integrated with both diagnostic and therapeutic moieties, and is often coupled with a ligand for active targeting The widely exploited nanomaterial for cancer nanomedicine research is a liposome which is loaded by doxorubicin drugs to make the first FDA approved nano drug Doxil™/Caelyx™ in vitro and 131I-TfR Ab displayed a feature of specific accumulation in tumor tissue in vivoTumor-tailored targeting approaches include active targeting. Drug-loaded nanoparticles modified with specific ligands can bind to receptors on the surface of tumor cells, leading to the accumulation of nanoparticles on the surface of certain cells + enhanced tumor targeting. Inside the core, a lipid-anchored near-infrared fluorophore DiR-BOA  serves as the model drug cargo and diagnostic moieties for optical identification. Results show that HPPS-mAb possesses the ability to reinforce targeting to tumor cells and achieves preferential recognition by TfR in a complex biological environment. Furthermore, the formed nanoparticles are capable of delivering their cancer therapeutic target siRNA payloads into TfR+ tumor cells to substantially mediate cell apoptosis. Therefore, the HPPS-mAb is a promising theranostic nanoplatform to enable amalgamation of therapy and diagnosis to many TfR+ tumors.In this study, we establish a TfR mAb  functionalized nanoparticle HPPS for TfR2.HepG2, U87, Hela, and MDA-MB-231 tumor cells  were cultured in DMEM complete growth medium  supplemented with 10% FBS  and 100 U/ml penicillin, 100 μg/ml streptomycin. CHO-hTfR (human TfR) cells which stably express hTfR-GFP and control CHOvec cells which stably express vector control The hybridoma 7579 was intraperitoneally inoculated into Balb/c (nu/nu) nude mice  to produce ascites for the preparation of monoclonal antibody against transferrin receptor (TfR mAb) as described before DiR-BOA-loaded HPPS was kindly provided by Professor Zhang Zhihong . HPPS was prepared firstly through a lipid emulsion film formation with DMPC , DSPE-PEG2000  maleimide, cholesterol oleate, and DiR-BOA. Then the lipid emulsion film was titrated with 18aa apoA-1 mimetic peptide (AP) solution to form spherical complete nanoparticle HPPS. Sulfhydryl modification of mAb and isotype  was achieved using thiolating reagent 2-Iminiothiolane  to crosslink with maleimide functionalized HPPS Transmission electron microscopy (TEM) was performed to determine the size dispersion and morphology of nanoparticles. The particle hydrodynamic diameter distributions were measured by dynamic light-scattering (DLS) using Zetasizer . The potential was measured using M3-PALS technology (Zetasizer Nano-ZS90). DiR-BOA concentration of nanoparticles was determined by a standard curve of DiR-BOA via detecting its absorption at 700 nm wavelength.CHO-hTfR cells were cocultured with nanoparticles (DiR-BOA 1 μM) for 30 min at 37 °C. The imaging was implemented through a Zeiss LSM710 confocal laser scanning microscopy  with excitation at 488 nm for EGFP (Enhanced Green Fluorescent Protein) and 633 nm for DiR-BOA. Images were acquired and analyzed using Zen-2009 software.5 cells  were incubated with different DiR-BOA concentrations of nanoparticles (10 μM~18 nM) for 30 min at 37 °C. After 3 times rinse, DiR-BOA+ cells were measured by flow cytometry  using APC-Cy7 color substitution excited at 633 nm. For HDL blockade assay, HepG2 cells were treated with 50 molar excess human HDL  for 15 min, then were incubated with HPPS-mAb or HPPS-isotype (DiR- BOA 10 μM). For apoptosis assay, HepG2 cells were stained with Annexin V FITC and PI  according to the manufacturer's specifications. Apoptotic cells were analyzed by flow cytometry.The incubation concentration consistency of HPPS and HPPS-mAb was referred to DiR-BOA determined by a standard curve. 3×107/ml cells (in 50 μl) mixed with matrigel . CHOvec cells were inoculated in the left hind flanks and CHO-hTfR cells in the right hind flanks. 7-14 days later, when tumor size (the largest diameter) of any side reached 10mm, tumor-bearing mice (four in each group randomized) were intravenously injected with nanoparticles at a DiR-BOA dose of 10 nmol via the tail vein. Whole-body fluorescence images were taken with a CRI Maestro in vivo imaging system at 12 h, 24 h post injection. The excitation wavelength of HPPS contained DiR-BOA was 700 nm and the emission filter was 750 nm long pass.All animal experiments were approved by the Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology in compliance with guidelines approved by Animal Care Committee. Balb/c nude mice  were subcutaneously inoculated with 1×10Cholesterol modified survivin-siRNA  consisted of the sense strand 5'-chol-AGCAUUCGUCCGGUUGCGCsTsT-3' and antisense strand 5'-GCGCAACCGGACGAAUGCUTsT-3'. Cholesterol-conjugated siRNA bearing a scrambled sequence (chol-si-NC) consisted of the sense strand 5'-chol-UUCUCCGAACGUGUCACGUsTsT-3' and the antisense strand 5'-ACGUGACACGUUCGGAGAATsT-3'. Chol-siRNA and HPPS-mAb were mixed at 1:1, 1:5, 1:10, 1:20, 1:30 molar ratio (siRNA: DiR-BOA) at RT for 30 min to obtain HPPS-mAb/siRNA. Then the HPPS-mAb/siRNA was loaded into 2% agrose gel containing nucleic acid dye. Naked siRNA and HPPS-mAb were mixed and run at molar ratio 1:5 using the same experimental condition.HepG2 cells were treated with HPPS-isotype/ siRNA and HPPS-mAb/siRNA for 48 h. Then cells were lyzed using Trizol  according to manufacturer's recommendation. The achieved mRNA was reverse transcribed to cDNA and amplified as previously reported HepG2 cell lysates were separated by 12% SDS- PAGE and transferred to polyvinylidene difluoride (PVDF) membrane. After blockade, membrane was probed with rabbit anti-survivin polyclonal antibody  followed by HRP-labeled goat anti rabbit antibody . Bands were detected using ECL kit . β-actin was set as loading control. Blank control referred to PBS.P < 0.05.The uptake of nanoparticles was analyzed by two-way ANOVA followed by LSD post hoc test. Survivin mRNA or protein expression was analyzed by one-way ANOVA followed by Bonferroni post hoc test. ζ-potential was analyzed by student's two-sided t-test using SPSS 17.0 statistical software . All values in the study were expressed as means ± SD. Differences were considered significant statistically as HPPS mainly contained lipids including DMPC, DSPE-PEG2000, CO (cholesterol oleate) and 18aa ApoA-1 mimetic peptide (AP). TfR mAb was isolated from ascites with purity of about 98%. The mAb was thiol-functionalized and conjugated to the surface of HPPS to obtain HPPS-mAb. DiR-BOA, a near-infrared fluorescent dye, which is a small hydrophobic molecule fluorophore amenable to bisoleoyl-based nanoparticle incorporation, has been loaded into the core of HPPS as a model functional cargo. A concise synthetic process was shown in Figure ζ-potential measurements showed that loading mAb on HPPS did not significantly change surface charge of the nanoparticles Figure B. DynamiThe molar concentration of DiR-BOA was employed to normalize the nanoparticle quantification. DiR-BOA dose referred to the concentration within the nanoparticles. To confirm the targeting specificity of HPPS-mAb, a plasmid encoding a hTfR-EGFP fusion protein was stably transfected into hTfR-negative CHO cells to establish CHO-hTfR cells, and the empty plasmid was treated the same to establish CHOvec cells  which indicated TfR targeting specificity of HPPS-mAb  on the left flank and CHO-hTfR (TfR+) on the right flank. HPPS-mAb and HPPS-isotype were injected via tail vein to compare the particles accumulation. Benefiting from its excellent penetrability, DiR-BOA was allowed for detection of nanoparticle internalization and tumor visualization. In Figure in vivo whole body images showed that loading with DiR-BOA in the core of nanoparticle, HPPS-mAb nanoparticles were observed to accumulate and DiR-BOA fluorescence signals were found to peak in CHO-hTfR tumor tissues as early as 12 h after systemic administration. The HPPS-mAb showed different distribution pattern between TfR+ and TfR- tissues. A direct superior accumulation of DiR-BOA meant that it was distributed to its target tumor tissues more quickly than to non-target tumor tissues. By contrast, HPPS-isotype rarely accumulated in bilateral xenografts at both time points. Taken together, these data demonstrated that HPPS-mAb can be targeted specifically to cancer cells through TfR at an early valuable node, and DiR-BOA can act as an imaging tool to diagnose cancer and to monitor therapy non-invasively and in real time.In order to investigate the + tumor in vivo, HPPS-mAb was incorporated by other hydrophobic therapeutic payloads to test its potential suitability for the targeted delivery of clinical relevant cancer therapeutics. Since the naked siRNA will be eliminated rapidly post intravenously injection as a result of kidney filtration and/or serum degradation, HPPS-mAb was loaded with cholesterol-modified survivin siRNA  Figure C.By above, HPPS-mAb could be a favorable nanoplatform to deliver therapeutic agents into tumor cells to achieve targeted tumor chemotherapy.in vitro and in vivo. Results manifested that in serum-rich conditions, TfR mAb grafted nanoparticles showed interaction with and internalization via TfR, and reduction of uptake in human TfR-negative CHO cells. The in vivo imaging showed the quick distribution of HPPS-mAb from blood to TfR+ tumor tissues, confirming targeted delivery of TfR mAb grafted nanoparticles in a complex biological environment. This enlightened us that this distribution mechanism could enable HPPS-mAb serve as potential diagnostic agent.Advancements in the development of nanotheranostics have made it possible for more accurate tumor detection, cancer staging, and real-time monitoring of cancer progression and therapeutic outcomes in vivo environment Except for being functionalized by targeting moieties to avoid normal tissue side effect, ideal nanocarriers should meet the following several points theoretically Data showed that the HPPS-mAb nanoparticles gained favorable tumor accumulation. However, when HPPS and HPPS-mAb were incubated with multiple tumor cells respectively, a gradient accelerating phenomenon leading to enhanced nanoparticle uptake was demonstrated, questioning whether a dual targeting cooperative mechanism existed. Given that HPPS mimics the functions of plasma-derived HDL not only in its pharmacokinetics but also in its targeting specificity against SR-BI which prominently expresses in the liver and steroidogenic cells of the adrenal and gonads especially in variety of tumors in vivo targeting imaging research. Whole body imaging showed that within 24 h, HPPS-mAb had a preferred accumulation in CHO-hTfR bearing xenograft while the HPPS-isotype owing the same nano-dimension performance showed no preferred orientation. We previously reported that radionuclide-labeled TfR-mAb accumulated and peaked in tumor tissue 24 h after systemic administration The isotype construction of HPPS-mAb benefited the HPPS-mAb in vivo. Researchers have focused their attention on siRNA biological property optimization and development of safe and effective transportation systems. Studies have been reported that the cholesterol modification of siRNA, 2'-fluoro substituted nucleic acids and phosphorothioate (PS) linkages usage would enhance potency of siRNA to resist nucleases in vitro and in vivo, HPPS becomes a safe, efficient siRNA carrier using the property of transporting intracellular active cancer agents to the cytosol without the involvement of endolysosomal trafficking Many nanoparticle-based delivery strategies have been developed for improving siRNA delivery The variations among individuals make a single nano-formulation not applicable for all kinds of tumors. The high and broad expression of TfR in various malignant tumors makes TfR mAb- functionalized nanoparticles applicable for as many tumor types as possible. And the carrier HPPS holds great relevance as they may represent biocompatible systems with relatively simple fabrication and modification. In our lab, this HPPS-mAb nanoplatform is developed to be incorporated with more other therapeutic payloads for tailored therapy for more cancer types.+ cancer cells. HPPS-mAb is a suitable theranostic nanoplatform for drug-release monitoring, imaging-guided focal therapy and post-treatment response monitoring.This study confirmed the TfR targeting, DiR-BOA imaging and effective drug release of HPPS- mAb nanocarriers in TfR


Omega-3 polyunsaturated fatty acids (omega-3 PUFAs), which are essential fatty acids that humans should obtain from diet, have potential benefits for human health. In addition to altering the structure and function of cell membranes, omega-3 PUFAs (docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), alpha-linolenic acid (ALA), and docosapentaenoic acid (DPA)) exert different effects on intestinal immune tolerance and gut microbiota maintenance. Firstly, we review the effect of omega-3 PUFAs on gut microbiota. And the effects of omega-3 PUFAs on intestinal immunity and inflammation were described. Furthermore, the important roles of omega-3 PUFAs in maintaining the balance between gut immunity and the gut microbiota were discussed. Additional factors, such as obesity and diseases , which are associated with variability in omega-3 PUFA metabolism, can influence omega-3 PUFAs–microbiome–immune system interactions in the intestinal tract and also play roles in regulating gut immunity. This review identifies several pathways by which the microbiota modulates the gut immune system through omega-3 PUFAs. Omega-3 supplementation can be targeted to specific pathways to prevent and alleviate intestinal diseases, which may help researchers identify innovative diagnostic methods. Gut microbes play vital roles in maintaining intestinal health . NutrienOmega-3 PUFAs including EPA, DHA, and ALA are essential fatty acids for animals . DPA is Accumulating evidence implicates the correlation between omega-3 PUFAs and gut microbiota. Omega-3 PUFAs can influence the gut microbial community; in turn, gut microbiota can also affect the metabolism and absorption of omega-3 PUFAs. However, the knowledge about the connections between omega-3 PUFAs and gut microbiota is limited. In adults, changes in the gut microbiota were observed after omega-3 PUFA supplementation [Omega-3 PUFAs affect the gut microbiome in three main ways: (1) modulating the type and abundance of gut microbes; (2) altering the levels of proinflammatory mediators, such as endotoxins (lipopolysaccharides) and IL17; and (3) regulating the levels of short-chain fatty acids or short-chain fatty acid salts.Enterobacteria, increasing the growth of Bifidobacteria, and subsequently inhibiting the inflammatory response associated with metabolic endotoxemia [Omega-3 PUFAs could directly modulate the diversity and abundance of the gut microbiota. Compared with sunflower oil, the dietary intake of fish oil exerted the greatest effect on the diversity of the intestinal flora . High leotoxemia .Bifidobacteria and Lactobacilli, in the distal intestine, thus affecting the distribution of the intestinal flora [Bifidobacterium [Bifidobacteria in the gut of male Sprague-Dawley rats [Bifidobacteria in the gut of the mice fed a high-fat diet [Akkermansia, improve the intestinal microenvironment, increase the intestinal mucosal thickness, improve the barrier function of the intestinal mucosa, and achieve weight loss by controlling the expression of genes related to fat metabolism [Studies using animal models show the association between fatty acid ingestion and changes in gut microbiota . Omega-3al flora . In addiacterium . Dietaryley rats . EPA andley rats  and incrfat diet , 22. Ometabolism .Escherichia, Faecalibacterium, Streptococcus, Sutterella, and Veillonella genera and the decrease of the abundance of the Bacteroides, Flavobacterium, and Oscillospira genera were detected in the IBD group after supplementation with omega-3 PUFAs, presenting the decreased F/B ratio [Omega-3 PUFAs could directly or indirectly alter the balance of gut microbes, contributing to the occurrence and development of multiple diseases . Omega-3/B ratio . Further/B ratio . In summOmega-3 PUFAs also can modulate gut microbes through inhibiting the production of proinflammatory mediators or promoting the production of anti-inflammatory mediators. In some cases, LPS passes through the intestinal wall, particularly when the barrier is destroyed, causing further damage . The incκB pathways induced by LPS. Incubation of macrophages with omega-3 PUFAs reduces MAPK kinase activity induced by LPS and decreases the expression of proinflammatory mediators, such as TNF-α [The consumption of omega-3 PUFAs inhibits the LPS-induced production of proinflammatory cytokines in human blood monocytes , relieveas TNF-α . Omega-3as TNF-α . Interleas TNF-α .Salmonella-infected mice significantly increased the SCFA content, thereby altering the gut microbiota and favoring host resistance to pathogens [Blautia, Bacterioides, Roseburia, and Coprococcus, was observed [Omega-3 PUFAs can also affect gut microbes through increasing the content of SCFAs. Omega-3 PUFAs exert a positive effect through restoring the microbiota composition in individuals with various diseases and increasing the production of anti-inflammatory compounds, such as SCFAs . Butyricathogens . In one observed . Increasobserved , 45.The effect of omega-3 PUFAs on the gut microbiota may be a main contributor to the health benefits of omega-3 PUFAs. Omega-3 PUFAs are mainly absorbed in the gut, where some microorganisms can directly utilize omega-3 PUFAs and produce numerous small molecules. Studies have highlighted the changes in the gut microbiota after omega-3 PUFAs supplementation . FurtherBacillus proteus or Lactobacillus plantarum, convert the omega-3/omega-6 PUFA precursors ALA and LA into CLA (conjugated linoleic acids) and CALA (conjugated α-linolenic acids), respectively, which are then further hydrogenated to saturated fatty acids , thereby reducing PUFA composition [Omega-3 PUFAs could directly affect the gut microbiota, and correspondingly, the gut microbiota could directly or indirectly modulate the absorption, bioavailability, and biotransformation of omega-3 PUFAs and further influence the imbalance of PUFA intake and its function. Gut microbes produce PUFA-derived metabolites, which may be novel active metabolites . As showposition . PUFA-deposition .Bifidobacterium modulates fatty acid metabolism or fatty acid uptake by the intestinal epithelium, but the mechanism underlying the association between Bifidobacterium and the absorption of omega-3 PUFAs was not elucidated [Bifidobacterium in the gut. An increase in the relative abundance of Bifidobacterium in the gut via the administration of probiotics or prebiotics also increases the blood omega-3 PUFA levels, which is beneficial to our health, such as preventive and therapeutic effects on cardiovascular diseases and affective disorders.The intestinal flora affects host health or nutrition-related diseases through regulating the digestion and absorption of PUFAs . The maiucidated . InteracBifidobacterium, Roseburia, and Lactobacillus in the mouse intestinal tract [Bifidobacterium and Lactobacillus [Bifidobacterium may be the main genus of bacteria that modulates the utilization of omega-3 PUFAs by microorganisms. Further studies are needed to explore the relationship between Bifidobacterium and omega-3 PUFAs.The effects of gut microbes on the metabolism and absorption of omega-3 PUFAs may be mediated by SCFAs. Omega-3 PUFA supplementation induces a reversible increase in the abundance of several SCFA-producing bacteria, containing al tract . In micebacillus . Based oα, IL-1β, IFN-γ, and IL-6), and aberrant changes in the microbiota composition . Omega-3 PUFAs have been shown to effectively counteract these adverse effects [Omega-3 PUFAs can improve intestinal immunity. Omega-3 PUFAs could reduce intestinal epithelial cell damage caused by LPS, sodium dextran sulfate, or hydrogen peroxide and increase intracellular mitochondrial activity and cell membrane integrity . Stress  effects .κB-mediated inflammation or attenuate the phosphorylation of MAPKs, subsequently reducing the transcription of inflammatory molecules [Bacillus species. The ingestion of omega-3 PUFAs inhibits LPS-induced proinflammatory cytokine production in human blood monocytes [Omega-3 PUFAs modulate intestinal immunity through three main mechanisms. First, omega-3 PUFAs reduce the release of membrane phospholipid arachidonic acid (AA) by reducing the intracellular AA content or by inhibiting phospholipase activity . Second,olecules . Finallyonocytes . Omega-3Accumulating evidences revealed omega-3 PUFAs, primarily EPA and DHA, suppress inflammation and exert a beneficial effect on a variety of inflammation-related diseases, such as inflammatory bowel disease, rheumatoid arthritis, asthma, cancer, and cardiovascular diseases . PUFAs sκB signaling pathways, (2) reducing the production of precursors that cause inflammation, and (3) altering the mechanism regulating the expression of inflammation-related genes (Omega-3 PUFAs may reduce inflammation through three main pathways: (1) mediating immune cell activation through the MAPK and NF-ed genes .κB, activator protein-1, and oxidative stress pathways or through increasing the activation of PPARγ or GPR120. Given the proinflammatory effects of several MAPKs, particularly extracellular signal-related kinases and c-Jun N-terminal kinase (JNK), the inhibition of specific MAPKs is a prospective mechanism by which omega-3 PUFAs block or reduce intestinal inflammation. Omega-3 PUFAs maintain intestinal health by reducing oxidative stress and NF-κB-mediated inflammation in immune cells and intestinal cells [κB signaling by activating peroxisome proliferator-activated receptor (PPAR)-γ [Omega-3 PUFAs reduce inflammation by decreasing the activation of proinflammatory MAPK, NF-al cells . Omega-3(PPAR)-γ , 62.γ levels in the intestine due to ischemia and reperfusion injury [Another possible mechanism is the suppression of inflammation through the activation of GPR120, an omega-3 fatty acid-activated receptor expressed in white adipose tissue (WAT) and bone marrow-derived dendritic cells and macrophages , 64. Forn injury . Thus, tn injury .The consumption of a diet rich in omega-3 PUFAs has been reported to protect intestinal cells from inflammatory damage that leads to IBD and to activate immune cells by reducing the production of proinflammatory eicosanoids. Omega-3 PUFAs may also exert their anti-inflammatory effects through incorporation into the plasma or phospholipid membranes of immune cells or intestinal mucosal tissues in human and rodent models . FurtherκB, thereby reducing the expression of inflammatory genes and activating the anti-inflammatory transcription factor PPARγ [Some of the beneficial effects of PUFAs are attributed to changes in the fatty acid composition of the membrane and subsequent alterations in hormone signaling. Omega-3 PUFAs disrupt lipid rafts and inhibit the activation of the proinflammatory transcription factor NF-or PPARγ . The fator PPARγ . PUFAs ror PPARγ , 74.β-oxidation, reducing hepatic inflammation and oxidative stress, and promoting intestinal homeostasis, suggesting that omega-3 PUFAs may be promising treatments in the management of alcoholic liver disease (ALD) [α (TNFα), interferon γ (IFNγ), and IL-4. In addition, dietary omega-3 PUFAs, which affect intestinal integrity, have been shown to reduce clinical colitis and colonic immunopathology by improving epithelial barrier function in animal models [Omega-3 PUFAs also alleviate alcoholic steatosis and alcohol-induced liver injury through various mechanisms, including reducing adipose tissue lipogenesis and lipid mobilization, enhancing mitochondrial fatty acid se (ALD) . EPA andl models . In addil models .κB, TNF-α, and IL-10). This diet also increased the amount of omega-3 PUFAs in the mucosa and decreased the ratio of omega-6/omega-3 PUFAs. In addition, the omega-3 PUFA-enriched diet decreased the abundance of pathogenic spirochaetes in the colonic digestive tract and increased the abundance of Actinomycetes, Blautia spp., and Bifidobacteria [Bifidobacteria [PUFAs simultaneously modulate the gut microbiota and immunity. Piglets fed omega-3 PUFA-enriched diets exhibit an increase in systemic and intestinal immunity, as evidenced by increased plasma concentrations of immunoglobulin G, decreased numbers of CD3+CD8+ T lymphocytes, and downregulated expression of intestinal genes  and butyric acid-producing bacteria [Omega-3 PUFAs also maintain host immunity by maintaining the balance between beneficial and harmful bacteria. A decrease in beneficial bacteria leads to a weakened intestinal resistance to harmful bacteria, resulting in a strong activation of proinflammatory signaling pathways, such as LPS-producing bacteria activate the NF-ytokines . Variousbacteria .Staphylococcus aureus (S. aureus) skin infections are inconsistent with the findings from human studies suggesting a protective effect of enhanced Treg function on S. aureus infection [Omega-3 PUFAs may modulate immune responses through several potential mechanisms. Primarily, omega-3 PUFAs directly modulate systemic immunity by altering the phospholipid membranes of immune cells, inhibiting omega-6-induced inflammation, downregulating inflammatory transcription factors, or serving as precursors of anti-inflammatory lipid mediators. The intestinal microbiota in the offspring of mice fed high-omega-3 diets was altered, with a moderate increase in the levels of the anti-inflammatory cytokine IL-10 in both the colon and spleen . Increasnfection . Finallynfection .Many factors can affect omega-3 PUFA-microbiome-host immunity interactions, containing obesity, cancer, genetic disorders, and metabolic diseases. Omega-3 PUFAs may interfere with the development of obesity by modulating the gut microbiota and influencing the function of white adipose tissue . SupplemBifidobacterium).Obesity is associated with low-grade systemic inflammation. The consumption of a high-fat diet modulates the gut microbiota to substantially increase intestinal permeability, leading to LPS absorption and metabolic endotoxemia that triggers inflammation and metabolic disorders . In partObese patients exhibit impair intestinal immunity due to a reduced gut microbial diversity, and metabolic pathway alteration leads to the level of DHA and EPA decreased, which can be alleviated by supplementation with omega-3 PUFAs . In addiLactobacillus rhamnosus to NAFLD mice for 8 weeks increases the abundance of beneficial bacteria in the distal small intestine and decreases portal alanine aminotransferase activity, thereby reducing the symptoms of NAFLD [Increased lipogenesis, hyperlipidemia, and increased fat deposition contribute to NAFLD development. NAFLD is characterized by triacylglycerol accumulation in hepatocytes (steatosis), which may progress to inflammation, fibrosis, and cirrhosis (steatohepatitis). Numerous studies have implicated the gut microbiota in the development of NAFLD , as it sof NAFLD .P < 0.0001) [Meanwhile, omega-3 and omega-6 PUFAs (omega-3/omega-6 PUFAs) have been linked to NAFLD , 92. The 0.0001) . In rats 0.0001) .β-catenin nuclear translocation [Omega-3 PUFAs are important lipids that participate in many pathological processes related to tumor occurrence and development by relieving inflammation . Omega-3location . While oOne possible explanation is that the modulation of the intestinal microbiota may contribute to the cancer-preventative properties of omega-3 PUFAs. Free feeding of EPA on mice with colon cancer increased the abundance of lactic acid-producing bacterial species in the gut . PatientStaphylococcus aureus produces enterotoxins in the human intestinal tract that wreak havoc on the human gut, causing symptoms such as vomiting and diarrhea [Staphylococcus aureus, and DHA and EPA have been used clinically as topical agents to treat skin lesions caused by Staphylococcus aureus [Citrobacter is a bacterium present in the intestinal tract of mice that promotes the proliferation of other pathogenic bacteria in the intestinal tract and causes gastrointestinal disease. An experiment conducted using mice with colitis showed that the administration of omega-3 PUFA-rich diet for 3 weeks altered the phospholipid composition of the intestinal cell membrane, reduced local inflammation, and reduced the production of proinflammatory cytokines and chemokines, thereby reducing colonic damage [Clostridium perfringens (a bacterium associated with IBD) and increasing the amount of Lactobacillus spp. and Bifidobacterium spp. with anti-inflammatory properties [Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pneumoniae and reduced the incidence of pneumococcal infections in the elderly [Omega-3 PUFAs may play a key role in the host defense against infections by limiting excess inflammation and enhancing the immune response , but bacdiarrhea . Omega-3s aureus . Citrobac damage . The conoperties , 105. Th elderly . The speWe reviewed the interactions among PUFAs, gut microbes, and host immunity. Based on accumulating evidence, omega-3 PUFAs  exert profound effects on the intestinal microbiota, the host-microbiome interaction, and interactions between the host immune system and gut microbiota. Accordingly, the gut microbiota modulates the absorption and metabolism of omega-3 PUFAs and directly or indirectly modulates subsequent physiological and immune responses in the host. In previous studies, researchers focused on the trends in the host digestion and absorption of omega-3 PUFAs, while the effects of gut microbes on omega-3 PUFAs have often been neglected. Therefore, further comprehensive studies about the effects of omega-3 PUFAs on gut microbes and gut immunity will be meaningful. Likewise, we also must determine which gut microbes, which type of omega-3 PUFAs, or which pathways affect gut microbial homeostasis and host immunity.Factors such as obesity and diseases are associated with host gut microbes, gut immunity, and omega-3 PUFAs. Omega-3 PUFAs modulate gut immunity by acting on gut microbes. In addition, omega-3 PUFAs are a feasible approach to maintain gut health. However, the composition of the gut microbes is complex, and simply using one substance will not be an effective method to solve these problems; individualized treatments for patients should be developed.


Background: The COVID-19 pandemic has changed the health security of older adults. Few have examined how older US veterans have reacted and coped with the COVID-19 pandemic. We aimed to identify changes in physical health and their differential impact by rurality of older veterans. Method: Participants were veterans (aged ≥ 62 years) and their cohabitants, living in the Pacific Northwest, enrolled in the Collaborative Aging Research using Technology (CART) initiative. Daily step counts via actigraphy were collected from January 1st to July 8th, 2020. COVID-19 time periods were determined by stay-at-home orders issued on March 13th, 2020. Generalized estimating equation models were used to examine changes in physical activities associated with COVID-19 time periods and rurality indicated by the rural-urban commuting area score. Results: A total of 102 participants were included in the analysis . Daily average step counts were 2318 and 3012 before and after COVID-19 stay-at-home orders . After controlling for covariates, participants living in large rural  and small/isolated areas  walked more than those living in urban areas after COVID-19 stay-at-home orders. Conclusion: Older adults cope differently during the COVID-19 pandemic based on rurality, with those living in large rural and small/isolated rural areas have an increased physical activity. Reasons for increased step counts  require further investigation. This result demonstrates the potential utility of real-world monitoring to objectively inform interventions for COVID-related secondary health changes.


Epstein-Barr virus (EBV) or human herpesvirus 4 (HHV-4) is a ubiquitous human oncogenic virus, and the first human virus found to express microRNAs (miRNAs). Its genome contains two regions encoding more than 40 miRNAs that regulate expression of both viral and human genes. There are numerous evidences that EBV miRNAs impact immune response, affect antigen presentation and recognition, change T- and B-cell communication, drive antibody production during infection, and have a role in cell apoptosis. Moreover, the ability of EBV to induce B-cell transformation and take part in mechanisms of oncogenesis in humans is well known. Although EBV infection is associated with development of various diseases, the role of its miRNAs is still not understood. There is abundant data describing EBV miRNAs in nasopharyngeal carcinoma and several studies that have tried to evaluate their role in gastric carcinoma and lymphoma. This review aims to summarize so far known data about the role of EBV miRNAs in altered regulation of gene expression in human cells in EBV-associated diseases. Herpesviridae, subfamily Gammaherpesvirinae, and genus Lymphocryptovirus [Epstein-Barr virus (EBV) or human herpesvirus 4 (HHV-4) is a ubiquitous human oncogenic virus that belongs to the family ptovirus . Since iptovirus ,3,4,5. Aptovirus . Howeverptovirus . Subsequptovirus ,8.MicroRNAs (miRNA) are small, non-coding RNA molecules of cellular or viral origin that consist of 18–22 nucleotides and play an important role in regulation of gene expression. Consequently, they affect key events in cell biology such as proliferation, apoptosis, and lipid metabolism. By binding to messenger RNAs (mRNAs), miRNAs induce degradation of mRNAs or inhibition of translation, thus reducing levels of expression of target genes. Virus-encoded miRNAs (v-miRNAs) are considered an important non-immunogenic tool for post-transcriptional regulation of both host and viral gene expression in infected cells . The majEcoR I and BamH I fragments gathered after sonification [BamH I fragment H rightward open reading frame 1 (BHRF1) mRNA. It produces three miRNA precursors  and, subsequently, four mature miRNAs [EBV genome is a double-stranded DNA molecule that consists of 175 kbp containing nearly 100 genes and coding for 44 microRNAs ,14,15. Ification . Today, fication . The fire miRNAs . The sece miRNAs ,19 , while BART miRNAs are transcribed in all latency stages . DespiteAlongside immune evasion strategies mediated by host miRNAs and viral glycoproteins, EBV miRNAs are another means by which the virus successfully avoids effector mechanisms of the host’s immune system . The maiEBV interferes with efficient initiation of innate immune response at the very first step, e.g., by targeting expression of pattern recognition receptors (PRR). The impaired expression of PRR affects subsequent signal transduction as well as cytokine synthesis . The twoThe role of EBV miRNAs in the evasion of NK-cell-mediated responses has been analyzed in nasopharyngeal carcinoma cells in vitro. Interaction between the natural killer group 2 member D (NKG2D) receptor on NK cells and major histocompatibility complex class I chain-related peptide A (MICA) is considered the key step in the recognition and killing of cancer cells. Expression of MICA in NPC is positively regulated by transforming growth factor β-1 (TGF-β1). Wong et al. (2018) showed that miR-BART7 reduces the expression of TGF-β1 in NPC cells and impairs the NK-cell-mediated recognition of virus-infected cells  Figure .Immune evasion strategies employed by EBV also target NLR family pyrin domain-containing 3 (NLRP3) inflammasomes, which are responsible for inflammatory responses mediated by IL-1β and IL-18 upon recognition of viral antigens. The activity of inflammasomes is targeted directly at the level of NLRP3 expression (via miR-BART15), or indirectly, by targeting the IL-1 receptor itself (via miR-BHRF1-2-5p) ,35 Figu.miR-BART1, miR-BART2, miR-BART10, and miR-BART22 suppress efficient CD8+ T-cell-mediated antiviral immune response by targeting IL-12, a pro-inflammatory cytokine responsible for differentiation of naive type 1 helper T-cell (Th1 cells) to mature Th1 cells, increased synthesis of IFN-γ, activation of NK- and T-cells as well as inhibition of angiogenesis ,37. In aEBV miRNAs impair mechanisms of specific immunity by affecting adequate antigen recognition at the level of antigen processing , transport of processed antigenic peptides to major histocompatibility complex (MHC) molecules (by targeting peptide transporter subunit TAP2), and antigen presentation (reduction of lymphocyte antigen 75 expression on dendritic cells) ,40,41,42T-cell-mediated immunity can maintain long term immune control over EBV replication  while clinical consequences associated with EBV infection in persons with impairment of T-cell development or function are shown to be very severe. This suggests that virus-specific T-cell responses represent the main means of protective immunity in EBV infection . EBV miRT-bet belongs to the T-box family of transcription factors that are the main enhancers of the Th1 differentiation pathway and subsequent Th1 cell-specific IFN-γ synthesis, which are important for efficient antiviral immunity. Inhibition of T-bet translation (with subsequent suppression of p53) by EBV miRNAs, originally shown in invasive nasal NK/T-cell lymphoma cells, is supposedly associated with the inhibition of Th1 differentiation pathways. As a result, the control of EBV replication is less efficient .However, T-bet also regulates transcriptional networks that are common among other types of immune cells (including dendritic cells and innate lymphoid cells) and is currently considered to have an important role in bridging innate and adaptive immunity  .In addition, T-bet acts as a selective repressor of transcriptional pathways associated with type I IFNs subsequent to IFN-γ-induced signaling ,45. TherEBV association with development and progression of malignant tumors is well known, especially its frequent infection of lymphoma and carcinoma cells. Nevertheless, the role of EBV miRNAs in tumorigenesis has only recently come into focus.EBV miRNAs have been linked to development and progression of various types of lymphoma, such as acquired immunodeficiency syndrome-related diffuse large B-cell lymphoma (AIDS-related DLBCL), EBV-positive DLBCL, Burkitt lymphoma (BL), nasal NK/T-cell lymphoma (NNL), Hodgkin’s lymphoma (HL), posttransplant lymphoproliferative disorder (PTLD), and others .miR-BHRF1-3 was found to be highly expressed in AIDS-related DLBCL compared to EBV-positive BL and EBV-negative DLBCL . miR-BHREBV-miRNAs belonging to BART clusters were found in pCNS PTLD, but with varying expression of different BART miRNAs . FurtherThe presence of diverse EBV miRNAs in different types of lymphoma was analyzed not only in patient samples, but it was also researched through cell line-based studies. In BL41/95 cell line, derived from BL, BHRF miRNAs were detected . The stuEBV is also known to be associated with the development of NPC and GC. Recently, the role of EBV miRNAs was thoroughly studied in these malignancies. BART miRNAs were first found in NPC, xenografted and propagated in nude mice ,53, and Generally, it is believed that BART miRNAs in carcinoma act in synergy or obtain significant effects by combining their individual activities, in cooperation or competition with cellular miRNAs. It was shown that BART miRNAs target numerous transcripts of different genes, thus deregulating various downstream molecules and signaling pathways . ConsequBART miRNAs also modulate host cell pathways by mimicking cellular miRNAs. Although this feature is still not explored enough, several BART miRNAs were shown to have “seed” sequences similar to those of cellular miRNAs: miR-BART5 compared to miR-18a and miR-18b ,79, miR-Overall, BART miRNAs are highly expressed in NPC and GC types associated with EBV infection where they act synergistically and have redundant activities, but also possibly differ in their target genes in different intracellular milieus.We envision that further detection of cellular processes affected and regulated by EBV miRNAs will contribute to better understanding of the role of viral non-coding RNAs in the development of virus-induced cancers in humans. In case of EBV, despite extensive research, there are currently no antiviral drugs or EBV-vaccines approved for use in humans. In years to come, better evaluation and understanding of the viral miRNA mechanisms might reveal new biomarkers and potential therapeutic targets.


Oncolytic virotherapy using oncolytic viruses with natural or engineered cancer-destroying capacities has emerged as a promising treatment concept in modern oncology. Rodent protoparvoviruses, in particular the rat H-1 parvovirus (H-1PV), have demonstrated their broad-range tumor-suppressive properties in both preclinical models and clinical studies. In addition to inducing selective tumor cell death, these viruses are also able to exert immunostimulating effects and reverse tumor-driven immune suppression. Parvovirotherapy holds therefore a potential for enhancing the efficacy of other cancer immunotherapies. The aim of this review is to provide an overview of all H-1PV-based combinatorial immunotherapeutic approaches against poor-prognosis human solid cancers that have been tested so far. Current challenges and future prospects of parvoviro-immunotherapy, notably parvovirus inclusion into various immunotherapeutic protocols against glioblastoma, pancreatic cancer, among other standard therapy-refractory solid malignancies, are also discussed in the light of H-1PV further clinical development.Resistance to anticancer treatments poses continuing challenges to oncology researchers and clinicians. The underlying mechanisms are complex and multifactorial. However, the immunologically “cold” tumor microenvironment (TME) has recently emerged as one of the critical players in cancer progression and therapeutic resistance. Therefore, TME modulation through induction of an immunological switch towards inflammation (“warming up”) is among the leading approaches in modern oncology. Oncolytic viruses (OVs) are seen today not merely as tumor cell-killing (oncolytic) agents, but also as cancer therapeutics with multimodal antitumor action. Due to their intrinsic or engineered capacity for overcoming immune escape mechanisms, warming up the TME and promoting antitumor immune responses, OVs hold the potential for creating a proinflammatory background, which may in turn facilitate the action of other (immunomodulating) drugs. The latter provides the basis for the development of OV-based immunostimulatory anticancer combinations. This review deals with the smallest among all OVs, the H-1 parvovirus (H-1PV), and focuses on H-1PV-based combinatorial approaches, whose efficiency has been proven in preclinical and/or clinical settings. Special focus is given to cancer types with the most devastating impact on life expectancy that urgently call for novel therapies. The rodent H-1 protoparvovirus (H-1PV)  of H-1PV-infected tumor cells (indirect immune cell stimulation): PVs are potent triggers of immunogenic stimuli through tumor cell ICD induction. Infected tumor cells release a spectrum of proinflammatory mediators, in particular chemo- and cyto-kines, and pathogen- and danger-associated molecular patterns , which are in turn capable of boosting the maturation and reactivity of distinct immune cell populations. This can be exemplified by H-1PV-infected human melanoma cells, which activate dendritic cell (DC) maturation through the release of heat shock protein 72 . In lineH-1PV infection of immune cells (direct immune cell stimulation): H-1PV infection of human immune cell subpopulations has been documented in various preclinical settings. Virus entry may take place in T, B, NK, DC and monocytic populations; however, infection is aborted at subsequent virus intracellular replication steps . AbortivH-1PV impact on tumor vasculature: It has been demonstrated that endothelial (precursor) cells may constitute direct targets for parvovirus-mediated toxicity. These cells sustain an abortive H-1PV infection in vitro. In animal models, virus treatment inhibits the growth of lymphatic endothelium-derived tumors (Kaposi’s sarcoma). Furthermore, recombinant propagation-deficient parvoviral vectors armed with angiostatic chemokines achieve significant reduction of vascular endothelial growth factor (VEGF) expression in Kaposi’s sarcoma cells . Given tThe above-outlined H-1PV potential for creating a proinflammatory immune environment and alerting the immune system to the presence of a tumor opens prospects for combining the virus with various immunomodulators or other therapeutic agents endowed with immuno-stimulating properties. This combinatorial approach is in particular promising for the treatment of human tumors that remain presently incurable and pose continuing research and clinical challenges. Pancreatic ductal adenocarcinoma (PDAC), glioblastoma, colorectal cancer (CRC) and melanoma are among those cancers, which are urgently calling for novel therapeutic strategies. H-1PV-based immunotherapeutic combinations are reviewed below, which aim at targeting these devastating malignancies.PDAC is the most common neoplasm of the pancreas and one of the most aggressive human cancers. It is characterized by quick progression, broad intraperitoneal dissemination  and frequent resistance to conventional treatments. PDAC is usually diagnosed at advanced stages, when surgical resection is either not feasible or inefficient, as most patients eventually suffer from local recurrence and metachronous metastasis . CurrentH-1PV is among the oncolytic viruses (OVs), which have promising potential for efficiently targeting pancreatic cancer. PDAC sensitivity to H-1PV-induced oncolysis was demonstrated in various preclinical models ,24. InfeAs gemcitabine is currently considered the gold chemotherapeutic standard in PDAC clinical management, the therapeutic efficacy of gemcitabine in combination with H-1PV was tested in a rat syngeneic orthotopic PDAC model. H-1PV administration to gemcitabine-pretreated animals led to significant tumor suppression and survival prolongation in comparison with the mock-infected or gemcitabine-only treated groups . These iOne study conducted in gemcitabine-treated PDAC patients revealed the ability of the drug to enhance T cell-mediated and DC-dependent host immune responses .In keeping with the aforementioned data, it was documented that gemcitabine therapy may promote naïve T cell activation in PDAC patients and enhance their responsiveness to specific vaccination or to other forms of immunotherapy .The understanding of gemcitabine immunoregulating effects as a complementary constituent of tumor cell toxicity was extended by the demonstration that this drug alleviates pancreatic cancer immune escape through NK cell cytotoxicity enhancement .Studies conducted in murine orthotopic PDAC models provided yet another insight into gemcitabine-mediated immuno-stimulation, namely by indicating that low chemotherapeutic doses selectively deplete effector/memory Treg cell populations. The latter has a strong impact on PDAC microenvironment, as Tregs usually form large intra-tumoral infiltrates and trigger local immune suppression ,29.Last but not least, in cancer models other than PDAC, gemcitabine enhances the efficacy of OV  therapy. This complementation is achieved through gemcitabine-mediated inhibition of myeloid-derived suppressor cell (MDSC) recruitment to the TME and acceleration of reovirus-induced antitumor T cell immune responses .Based on favorable preclinical data hinting at the potentiation of OV-induced antitumor effects in the presence of gemcitabine, a clinical trial, ParvOryx02 NCT02653313), was designed and conducted with the aim to provide a clinical proof-of-principle of the safety (and efficacy) of H-1PV + gemcitabine co-treatment. Patients with inoperable metastatic (at least one hepatic metastasis) pancreatic cancer were treated with H-1PV. The virus was first administered intravenously , and the remaining virus dose was then given intra-metastatically as single hepatic injection, followed by gemcitabine treatment 3, was de.Preclinical proof-of-concept was also obtained for another treatment combining H-1PV with the histone deacetylase (HDAC) inhibitor (HDACi) valproic acid (VPA) . HDACis Exert epigenetic regulation of various immune functions, e.g., attenuation of MDSC immunosuppressive effects .Induce the expression of MHC I-related chain A (MICA) and B (MICB) molecules, as well as of UL16-binding proteins (ULBPs) in human tumor cells, thereby triggering their enhanced recognition by NK cells , like H-Mediate the inhibition of macrophage migration inhibitory factor (MIF) expression through local chromatin deacetylation-based transcription targeting .It is noteworthy that besides its effects on tumor cell growth and OV oncotoxicity, VPA was reported to modulate the immune system, providing an additional possible interface for cooperation with OVs at the level of their intrinsic immuno-stimulating activity. VPA was indeed shown to:As a whole, the above data speak for the high translational relevance of VPA to the future development of PV-based combinatorial (immuno) therapies.In conclusion, two drugs that are available on the pharmaceutical market, i.e., gemcitabine (cytostatic) and VPA (antiepileptic), proved to be efficient in synergizing with H-1PV to suppress pancreatic cancer .Another combination with substantial potential for clinical development relies on the mutual complementation of H-1PV- and IFN-γ-mediated immune stimulation. It was shown that IFN-γ improves the vaccination potential of the virus and diminishes the development of peritoneal carcinomatosis in preclinical PDAC models. Concomitant intraperitoneal administration of both H-1PV and IFN-γ in these models led to extended animal survival correlating with enhanced peritoneal macrophage and splenocyte responses against tumor cells .Glioblastoma multiforme (GBM) is the most common and aggressive human primary brain tumor. Similar to PDAC, GBM patients experience a very poor outcome. The 5-year overall survival rate is very low, around 5.1% . GBM treBased on the so far unmet need for novel, more efficient treatments, GBM was among the preclinical tumor models most extensively studied in our laboratory. H-1PV capacity for selectively killing glioma cells through cytosolic activation of lysosomal proteases was first demonstrated in vitro . These rPursuant to the above-described preclinical findings, the ParvOryx01 trial (NCT01301430) in recurrent glioblastoma patients delivered the first clinical proof-of-concept for tumor-infiltrating lymphocytes (TILs) playing substantial role in H-1PV-mediated immunomodulation of GBM TME. Although ParvOryx01 primary objectives were to determine virus safety, tolerability, pharmacokinetics, shedding and maximum tolerated dose, the analysis of post-virus-treatment resected tumor tissues revealed the presence of prominent immune cell infiltrates . These iWe have previously shown that radiotherapy, one of the conventional first-line treatments in glioblastoma patients, sensitizes low-passage glioma cultures to H-1PV oncolysis. Pre-irradiation increases the susceptibility of these cells to virus infection. Interestingly, H-1PV achieves killing both radiation-sensitive and resistant glioma cells . Apart f®). This co-treatment was evaluated in a series of compassionate virus uses in recurrent glioblastoma patients. Bevacizumab is an anti-VEGF-A monoclonal antibody available in Europe since 2005 for the treatment of breast, lung, kidney, colon, ovarian and endometrial carcinomas. In 2009, bevacizumab was approved by the Food and Drug Administration (FDA) for application in glioblastoma patients [Another promising approach is the combination of H-1PV with bevacizumab , is presently at the leading edge of cancer immunotherapy. Although efficient in controlling various other solid tumors, immune checkpoint inhibitors (ICIs) frequently fail to achieve a significant response in glioblastoma patients . SeveralFirst clinical hints of improved antitumor effects of H-1PV virotherapy upon combination with checkpoint blockade were obtained through compassionate virus uses. A series of three patients with rapidly progressing recurrent glioblastoma were treated with H-1PV (two were irradiated prior to virus administration), followed by bevacizumab and the programmed cell death protein 1 (PD-1) inhibitor nivolumab. In addition, all patients received the HDACi VPA. This innovative PV-based multimodal strategy led to radiologically confirmed tumor regression accompanied by clinical improvement in all subjects 4 to 8 weeks after virus injection . An objeAltogether, the above data provide a strong impetus for further clinical development of H-1PV combinations with radiation and/or immunomodulators (in particular bevacizumab and ICIs) in the fight against glioblastoma.CRC is another major cause of cancer-related deaths worldwide. Although the implementation of early-detection screening programs has substantially improved the 5-year overall survival, prognosis for CRC patients with stage 4 metastatic disease remains poor . ImmunotMany tumor types, including CRC, overexpress the immune checkpoint cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and thus transmit inhibitory signals to T cells . This imCutaneous melanoma, also known as black skin cancer, is an aggressive tumor arising from the melanocytes. Over the past 10 years, melanoma has become a prototype for testing novel targeted therapies, first and foremost, immune checkpoint blockade. PD-1 inhibition has shown significant clinical success in controlling locoregional melanoma . HoweverIn order to investigate the immunological effects of H-1PV in combination with ipilimumab and/or nivolumab, a human ex vivo melanoma model was used by Goepfert et al. Similar to the observations made in CRC-derived cells , upregulPreclinical research and clinical experience have demonstrated the multimodal anticancer activity of the oncolytic parvovirus H-1PV. Two essential facets of H-1PV-induced tumor suppression consist of direct killing of malignant cells (oncolysis) and activation of cellular immune responses against the tumor. H-1PV infection, oncolysis and immune stimulation are interconnected, coordinated events, which cooperate towards multisided tumor elimination.Glioblastoma and pancreatic adenocarcinoma are among the most devastating human malignancies, characterized by resistance to current therapies, tendency to recurrence and an overall poor outcome. H-1PV has undergone clinical testing in two recently conducted trials, ParvOryx01 in glioblastoma and ParvOryx02 in pancreatic carcinoma. Virus excellent safety and tolerability, together with the capacity for gentle TME immune landscape proinflammatory modulation, provide a strong impetus for further H-1PV clinical development. It should, however, be noted that in the clinical setting, various patient-dependent factors may result in suboptimal antitumor effects. Large intra-tumoral tissue heterogeneity, emergence of tumor cells resistant to virus infection/killing, dominance of the highly immunosuppressive TME, hampered virus spreading, off-target infection and virus neutralization by antiviral antibodies are among the major barriers to efficient H-1PV-induced tumor elimination. While various other approaches  to H-1PV treatment optimization are currently under investigation, PV-based combinatorial therapies are considered as a particularly promising avenue that holds the potential of enhancing both oncolysis and immune-mediated tumor destruction. Combinations of the virus with other anticancer approaches, namely irradiation, chemotherapy (gemcitabine), epigenetic modulation (HDACi), angiogenesis regulation (bevacizumab) or immunotherapy (immune checkpoint blockade), were evaluated in both preclinical models and in cancer patients. The combinatorial H-1PV-based viro(immuno)therapeutic strategy was proven to achieve greater anticancer effects compared to individual agents alone. The synergistic boost was particularly pronounced in combinations including the HDACi VPA, bevacizumab or the PD-1 inhibitor nivolumab. Glioblastoma patients treated with this combination showed striking tumor remission and extended survival, notably after second or even third recurrence. These early clinical observations speak in favor of considering H-1PV inclusion into various immunotherapeutic protocols against glioblastoma and other poor-prognosis solid tumors .