Targeted delivery approaches for cancer therapeutics have shown a steep rise over the past few decades. However, compared to the plethora of successful pre-clinical studies, only 15 passively targeted nanocarriers (NCs) have been approved for clinical use and none of the actively targeted NCs have advanced past clinical trials. Herein, we review the principles behind targeted delivery approaches to determine potential reasons for their limited clinical translation and success. We propose criteria and considerations that must be taken into account for the development of novel actively targeted NCs. We also highlight the possible directions for the development of successful tumor targeting strategies. Targeted delivery strategies based on nanocarriers have immense potential to change cancer care but current strategies have been shown only limited translation in the clinic. Here, the authors survey the challenge, progress and opportunities towards targeted delivery of cancer therapeutics. However, only a small percentage of these NCs accumulate even in high-EPR xenografted tumors . This could be due to multiple physiological barriers ; some get physically “stuck” in the sinusoids of the liver and others are taken up by hepatocytes and Kupffer cells4.Approaches for targeted delivery of therapeutics in cancer typically involves systemic administration of therapeutics packaged in nanocarriers (NCs) or localized delivery of therapeutics to the diseased tissue. Encapsulation of therapeutic molecules  in NCs can improve their solubility and bioavailability, alter their bio-distribution, and can also facilitate entry into the target cell. “Passively” targeted NCs, which utilize the enhanced permeability and retention (EPR) effect7. Over 40,000 studies published in the last 10 years have focused on active targeting strategies and substantial progress has been made toward our understanding of how NCs interact with cells and tissues. However, we still have not been able to overcome the challenges presented by physiological barriers 10. However, actively targeted NCs must first reach the target to take advantage of this increased affinity and avidity. Efficient passive targeting is therefore a prerequisite for NCs designed to systemically target tumor specific-cells or the extracellular matrix (ECM). Our understanding of EPR-related phenomena and its effect on NCs’ accumulation and penetration into tumors is mostly based on fast-growing xenografted mice models with dense vasculature, which does not recapitulate the majority of solid tumors in humans11. Several approaches have therefore been suggested to augment  or bypass  the EPR effect in low-EPR tumors or tumors in secluded organs , respectively.Active cellular targeting strategies involve utilizing affinity ligands on the surface of NCs for specific homing, increased retention at the target site, and uptake by the target cellsAdditional obstacle toward maximizing the efficacy of NCs is pre-mature release of therapeutics. Several strategies have been devised to overcome this issue, including the development of stimuli-responsive NCs. However, these features add another level of complexity to the NC design.In this review, we critically discuss the challenges and reasons behind limited clinical success of targeted delivery approaches in cancer treatment, in-spite of the huge number of published reports demonstrating their therapeutic potential in pre-clinical models. We also propose the focus areas for future research that will enable successful clinical translation of promising strategies for targeted delivery of cancer therapeutics.12. Passive targeting of NCs occurs due to unique characteristics of solid tumors , which allow NCs to preferentially accumulate in the tumor. This phenomenon, first described in 1986 by Matsumura and Maeda, is known as the EPR effect14. A number of passively targeted NCs are currently in clinical use 7. Other than that, several other NCs, including EndoTAG-1, AZD2811, and CPX-1 have also demonstrated safety and/or therapeutic effects in clinical studies17. Although most of these NCs simply alter the pharmacokinetics, toxicological profile, or solubility of drugs, few have also shown significant survival benefits and improvement in therapeutic efficacy over the parent drug in clinical studies. One example is Abraxane™ (nab-paclitaxel) that demonstrated significantly higher response rates compared to standard paclitaxel in a phase III trial conducted in patients with metastatic breast cancer18. Similarly, CPX-351 (Vyxeos™)—a recent FDA approved liposomal formulation of cytarabinee–daunorubicin combination has showed an improved overall survival of 9.56 months compared to 5.95 months as observed with cytarabine and daunorubicin given in their free form in patients with newly diagnosed high-risk acute myeloid leukemia19. This is extremely encouraging for the field and opens up new opportunities in utilizing NCs as delivery vehicles for multiple drugs.Passively targeted NCs first reached the clinic 22 years ago with the approval of PEGylated liposomal doxorubicin (DOXIL™)20. The tumor periphery is less permeable than the hypoxic core, which suggests that NCs should extravasate more frequently at the core than the periphery. However, multiple studies indicate the opposite—intravenously administered NCs extravasate more frequently in the tumor periphery22. Furthermore, permeability is not the only limiting factor; NC extravasation has also been shown to be governed by perfusion, which displays both spatial and temporal heterogeneity within a tumor, adding another level of complexity to controlling NC extravasation23. Additionally, physiochemical properties such as size and shape also affect NC extravasation and accumulation25. Even NCs that do cross the vasculature and extravasate into the tumor are impeded by the interstitial tumor matrix, which forms a barrier to their deep penetration into the tumor tissue. Reduction in the particle size significantly reduces the diffusional hindrance, thereby improving its penetration into the interstitial matrix. For example, Wong et al. proposed a multistage approach wherein a gelatin particle 100 nm in diameter upon its extravasation into the tumor tissue is reduced to a 10 nm particle via degradation by tumor-associated matrix metalloproteinases (MMPs)26. Similar approaches using different NCs have also been reported by many other groups29. Although promising, these approaches have not yet been evaluated clinically.Therapeutic efficacy of passively targeted NCs is impacted by the heterogeneity of the EPR effect within and between different tumors. Variable endothelial gaps (ranging from one to hundreds of nanometers) result in non-uniform extravasation of NCs into the tumor30.The 50-nm NCs demonstrated the highest tumor penetration, most efficient uptake by tumor cells and slowest tumor clearance, resulting in highest tumor tissue retention integrated over time and highest efficacy against both primary and metastatic tumors in vivo. Similar findings correlating size of NCs to their biological effects have also been reported by multiple other groups. Other physicochemical properties of NCs, including shape, elasticity and surface charge can also impact the interaction of NCs with physiological barriers and tumor microenvironment and therefore can be crucial to optimize NC design and maximize their biological function. For example, the shape of NCs has been reported to be a crucial factor in determining their blood circulation, ability to marginate in blood vessels, and uptake by tumor cells and macrophages34. Elasticity of NCs can also impact their biological fate, including blood circulation and tumor uptake. Anselmo et al. demonstrated that softer nanoparticles (10 kPa) result in prolonged blood circulation compared to harder nanoparticles (3000 kPa) in vivo35. Overall, physiochemical properties of NCs can substantially impact their accumulation, retention, and penetration in tumors. However, Sykes et al. demonstrated that the optimization of physiochemical properties of NCs is specific to the target tumor’s pathophysiology, and therefore must be personalized to each tumor type and stage to maximize therapeutic efficacy36.The size and geometry of NCs has also been shown to impact their other interactions with physiological barriers and tumor microenvironment. For example, Tang et al. compared the biological profiles of silica nanoconjugates with three different sizes  through both experiments and mathematical modeling11. There is also limited patient-based experimental data on the EPR phenomenon itself and its effect on drug accumulation in the tumor site that translates into clinical efficacy11. Further investigation of the EPR in various human tumors and the development of better preclinical models is therefore essential for the design of NCs with better tumor penetration and therapeutic outcome10. Recently, Theek et al. investigated the correlation between tumor vascularization and EPR-based passive targeting in a subcutaneous tumor model37. Utilizing both contrast-enhanced functional ultrasound and computed tomography-fluorescence molecular techniques, they demonstrated heterogeneous accumulation of 10-nm near infrared-labeled polymeric NCs (pHPMA-Dy750) within and between the tumors (5%–12%). Similarly, Hansen et al. recently developed copper-64-loaded PEGylated liposomes and evaluated the EPR effect of these liposomes by microPET/CT imaging38. Evaluation of 11 dogs bearing spontaneous solid tumors revealed that while the EPR effect is a predominant feature in few solid tumors , resulting in high liposome accumulation, it could not be generalized to all solid tumors. In a more advanced study, Miller et al. showed that an FDA-approved 30-nm carboxymethyl dextran–coated magnetic nanoparticle (MNP) (ferumoxytol) could be used as a surrogate or companion particle to predict intratumoral transport, pharmacokinetics (PK), and distribution of a therapeutic NC based on poly-b-poly(ethylene glycol) (PLGA-PEG)39. Recently, Lee et al., utilized 64Cu-labeled HER2-targeted liposomes and PET/CT to quantify drug accumulation in 19 patients with HER2 positive metastatic breast cancer40. The peak liposomes accumulation was observed at 24–48 h and patients were classified based on 64Cu-liposomal lesion deposition using a cut-point that is comparable to a response threshold that was measured in preclinical studies. Patients with high 64Cu-liposomal lesion deposition were associated with more favorable treatment outcomes. These studies demonstrate that utilizing imaging techniques for evaluation and characterization of EPR could eventually enable clinicians to pre-select patients with high-EPR tumors who are likely to respond to passively targeted NCs, thus remarkably improving therapeutic outcomes.Our current understanding of EPR effectiveness is limited by the scarcity of data obtained using pre-clinical tumor models that accurately recapitulate solid tumors in humans. In fact, the most commonly used subcutaneous tumor xenografts are rapidly growing, resulting in very high-EPR tumors that could provide a false impression of the therapeutic benefit of NCs in therapies that rely on passive EPR-based targeting2. In absolute terms, this number seems small, raising serious questions about the efficiency of the EPR effect and strengthening concerns regarding treatment of low-EPR tumors. However, in relative terms, a delivery efficiency of 0.7% for NCs is substantially higher than the delivery efficiency for most of the conventional formulations of chemotherapeutics that are currently dominant in the clinic, including docetaxel, paclitaxel, and doxorubicin44. For example, a pre-clinical study by Vlerken et al. demonstrated delivery efficiency of 0.6% ID for paclitaxel-loaded NCs compared to 0.2% ID for free paclitaxel41. This is encouraging and clearly establishes the advantage of NCs for tumor targeted drug delivery. However, delivery efficiency of NCs can be further improved in absolute terms to maximize their therapeutic benefit. Augmenting EPR effects using angiotensin II-induced hypertension or heat-based vasodilation could be one solution, though either technique could complicate the clinical translation of NCs. Another potential and relatively translatable solution, especially for low-EPR tumors is the development of sophisticatedly engineered delivery systems that exploit non-EPR approaches for tumor targeting. For example, Xu et al. developed an injectable nanoparticle generator (iNPG) that does addresses the concerns with multiple physiological barriers45. Specifically, the iNPG is a discoidal micrometer-sized nanoporous silicon particle that can be loaded with drug polymer conjugates. Demonstrating tumor accumulation due to natural tropism and enhanced vascular dynamics, the iNPG releases the drug polymer conjugate, which self-assembles to form nanoparticles that are transported to the perinuclear region, thereby bypassing the drug efflux pump. iNPG showed greater efficacy in MDA-MB-231 and 4T1 mouse models of metastatic breast cancer compared to its individual components and other current therapeutic formulations. Development of such rationally engineered systems could significantly improve the delivery efficiency of NCs.A recent meta-analysis of pre-clinical data on NC-based delivery platforms for tumors published over the past 10 years suggested that a median of about 0.7% of the injected dose (ID) of NCs reaches the target tumors46. Huang et al. harnessed the inherent ability of T-cells to traffic throughout the lymphatic system by conjugating nanocapsules encapsulating the topoisomerase I drug SN-38 to the cell surface47. Cell-mediated delivery resulted in 90-fold increase of SN-38 concentrations in lymph nodes than free drug, administered systemically at 10-fold higher doses, and also prolonged median survival by 35 days with no evidence of toxicity. In addition to targeting low-EPR tumors, immune cell-mediated delivery of NCs can also elicit improved tumor accumulation in disseminated tumors and metastases. This could open new avenues for safer targeted delivery of immunomodulating molecules such as IFN-γ, which can promote differentiation of tumor-promoting M2 macrophages to anti-tumor M1 macrophages. Furthermore, using tumor infiltrating lymphocytes or CAR T-cells for targeted delivery of NCs loaded with immunomodulating agents might enable a synergetic dual-arm treatment, augmenting anti-tumor immune responses with tumor targeting, and/or modulation of immunosuppressive cells. However, this approach is limited to therapeutics with low toxicity to normal carrier cells.Cell-mediated delivery of NCs could be another EPR-independent approach to enhance tumor targeting in low-EPR tumors or certain metastatic tumor locations that are unreachable by passive targeting. This approach exploits the ability of certain cell types to home or migrate to such tumors49. However, Kirpotin et al. established that incorporation of targeting ligands on the surface of NCs increases their cellular internalization by the target cells without affecting overall tumor localization50. This was also confirmed by Shmidt and Wittrup, who developed a mechanistic model for better understanding and predicting the complicated relationships between molecular size, binding affinity, and tumor targeting51. Their model predicts that for NCs with diameters ≥50 nm active targeting does not significantly increase the tumor localization of NCs compared to non-targeted NCs. This is a major difference between actively targeted NCs compared to small molecules and antibodies.Active cellular targeting was developed as a complementary strategy to passively targeted NCs for improving tumor localization of NCs by increasing their targeting efficiency and increasing retention at the target site52. Multiple strategies have been tested to facilitate endosomal escape of NCs into the cytosol, such as pore-formation peptides and proteins, pH-buffering substances utilizing the “proton sponge effect” and fusogenic NCs53. However, endosomal escape of NCs in vivo remains extremely challenging. Another complexity resides in the endosomes recycling process. A study by Sahay et al. demonstrated that the vast majority (70%) of siRNA containing Lipid nanoparticles (LNPs) that were internalized underwent exocytosis53. Gilleron et al. recently investigated the intracellular fate of siRNA-containing LNPs in vitro and in vivo54. Utilizing quantitative fluorescence imaging and electron microscopy, they demonstrated that the endosomal escape of siRNA in hepatocytes occurs at very low efficiency (1–2%) and only when the LNPs reside in a specific compartment sharing early and late endosome characteristics54. This discovery emphasizes the importance of improving our understanding of the mechanisms of endosomal escape in different cell types to achieve improved cargo delivery efficiency, which in turn could yield better therapeutic outcomes with fewer off-target effects. Furthermore, this could have wide implications in terms of reducing the toxicity and economic concerns.Since targeting ligands on NCs facilitate their uptake by target cells, this strategy has been utilized to enhance the delivery of high molecular weight molecules  to their target cells. These molecules are sensitive to enzymatic degradation and cannot cross the cell membrane to reach their active site within target cells. However, actively targeted NCs aimed at macromolecule delivery face additional physiological barriers arising from their interaction with their target cells. One of the major barriers is escaping the endocytic pathway. After endocytosis, NCs are directed to subcellular locations by intracellular trafficking mechanisms, which may have a detrimental effect on the fate of the NCs. For example, NCs that are internalized through clathrin-mediated endocytosis  would enter the degradative pathway and eventually be degraded in lysosomesTraditionally, choosing a target for actively targeted NCs is based on “classical” or disease markers . While these targets are clinically relevant for therapeutic monoclonal antibodies, they might not be suitable for efficient NC internalization and escape from the endosome when the therapeutics need to be delivered intracellularly . Therefore, targets should be screened not only based on their expression levels, but also by their ability to enable rapid internalization and efficient endosomal escape. Overall, the design of actively targeted NCs as delivery vehicles is complex, since factors such as the NC’s architecture, ligand conjugation chemistry, and the choice of the ligand contribute to the efficacy of the delivery system.55. The majority of active, cell-specific NCs target a single cell-surface receptor on tumor cells, thus disregarding tumor heterogeneity and promoting selection toward the survival of resistant clones. Consequently, current treatment usually results in apparent partial or complete responses, which in most cases followed by a resistant tumor relapse and mortality56. Ultimately, we argue that the development of a novel class of NCs with versatile targeting moieties or combining several NCs with different targeting moieties is crucial for improving efficacy.Additional complexity resides in the vast heterogeneity within and between tumors and the presence of tumor and metastasis supporting stroma 57. In this study, they utilized a lipidated single chain Fv-based linker platform that can self-assemble into lipid-based NCs and bind IgGs from a certain isotype. The incorporation of the linker to the lipid NCs by self-assembly resolves the complexity of the chemical conjugation of antibodies to NCs as well as the need for removal of unbound antibodies. Furthermore, the use of linkers that can bind different targeting moieties can enable the development of a single versatile NC platform that can be redirected to different cellular targets simply by mixing with different targeting moieties. Such strategies might enable a real breakthrough in overcoming the challenges posed by tumor heterogeneity, without making the manufacturing process of NCs more complex.Conjugation of targeting ligands to NCs results in a relatively complex manufacturing process compared to passive NCs, due to the additional steps of chemical synthesis and purification. Although this has not been the foremost limiting factor in clinical translation of actively targeted NCs, it does pose a significant challenge toward bench to bed translation of this approach due to more quality control steps, increased cost, and longer timelines. Recently, Kedmi et al., presented a possible solution to simplify the manufacturing process of actively targeted NCs and control the targeting agent orientation of the NCs’ surface58. For example, the formation of protein corona could affect many properties of NCs including size, stability and surface properties that define their cellular uptake, intracellular trafficking, pharmacokinetics, biodistribution, and toxicity60. In addition, the protein corona might mask the NC’s targeting ligands, compromising the targeting capabilities61. There have been numerous in vitro studies on NCs–protein interactions; however, the correlation of NCs–protein interaction in vivo with aforementioned biological responses are not yet extensively evaluated. NCs–protein interactions also restrict NCs’ circulation life due to the promotion of opsonization and recognition by MPS. However, it is worth noting that in tumors with a large volume of blood flow, NCs may not require a long circulation time to achieve effective tumor accumulation. There may also be undesirable interactions between NCs and the immune system, which in turn could lead to immunostimulation or immunosuppression. For example, the ability of some cationic NCs to activate toll-like receptor (TLR) signaling is well established62, but we know little about the immunological interaction of NCs with intracellular receptors such as the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and the inflammasome.Many factors including the route of administration and the coating formed by serum proteins and opsonins  have been shown to affect the targeting efficiency of NCs Fig. , as well64. However, there is increasing evidence that the “stealthness” of PEGylated NCs is hampered by unwanted adverse effects such as complement activation, which may result in hypersensitivity reactions and even anaphylaxis66. The inadvertent recognition of NCs by the immune system may elicit a multilevel immune response, eventually leading to toxicity, adverse effects in the host and/or lack of therapeutic benefit. Thus it has become crucial to predict these reactions with the NC surface at the molecular level using appropriate pre-clinical models. This task becomes more complex in the development of actively targeted NCs. For example, when decorated with full-length IgG molecules as a targeting moiety, these NCs are recognized by the MPS via Fc receptors. This challenge could be overcome by either using smaller antibody fragments ’2, etc.) or other homing molecules . Functionalizing NCs with “self” markers is another strategy to limit the interactions of NCs with serum proteins and immune system during systemic circulation70.Various surface-coated molecules have been utilized to provide “stealth” properties to NCs during systemic circulation; the most common being PEGOverall, nano–bio interaction is an extremely important research area in the development of NCs. In addition to NC–protein interaction and NC–immune system interaction during blood circulation, NC–ECM and NC–cell interaction are also crucial and have been discussed in the previous sections. With continuous improvements in our understanding of nano–bio interactions, more effective strategies could be developed to improve the targeted delivery of therapeutics to tumors.73 and multi-layered NCs for tissue and cellular targeting76). Tools to evaluate the interaction of NCs with the immune system are already being developed  and will enable better prediction of NC-mediated toxicity in design of future clinical trials.Passively targeted NCs, which rely solely on the EPR effect, may be insufficient to achieve efficient tumor targeting. We need more systematic studies to understand the interaction of NCs with physiological barriers, and the cues identified from those should be used to develop more sophisticated strategies. Utilizing natural tropism of certain cells toward specific tissues and harnessing tumor infiltrating lymphocytes are other strategies to improve NC accumulation in tumors. Recent advances in NC engineering have enabled overcoming some of the physiological barriers  production and add significantly to the cost of the therapy. SynerGene Therapeutics’ SGT-94, a NC made from cationic liposomes encapsulating tumor suppressor gene RB94 and decorated with an anti-transferrin receptor (TfR) single-chain antibody fragment is an example of an actively targeted NC with a relatively simple design; it is now in a Phase I trial in patients with genitourinary tumors.Despite multiple complexities, one major advantage of actively targeted NCs is their ability to target disseminated locations throughout the body, potentially revolutionizing the treatment of hematological malignancies  and of metastatic lesions in which EPR does not play a role. However, to ensure clinical success of actively targeted NCs, significant efforts and criticism should be exercised in the design of preclinical tumor models to ensure better recapitulation of the human disease, including both solid and hematological malignancies.Additionally, for both passive and active targeting strategies, the development of companion diagnostic imaging technologies to evaluate the targeting efficiency of NCs is crucial. Pre-selection of suitable patients and tailoring treatments to specific patients will improve tumor accumulation, treatment efficacy, and therapeutic outcomes. Furthermore, excluding unsuitable patients will reduce the incidences of adverse reactions and unnecessary treatments, reducing the expenses of both governmental health authorities and health insurance companies.77. In this work they used antibody-functionalized gold NCs with either single or two different types of antibodies. Although as expected, the NCs harboring two antibodies on their surface showed moderate improvement in in vitro targeting compared to NCs with single antibody, the opposite phenomenon was observed in vivo. These results highlight the concept that targeting ligand density should be optimized both in vitro and in vivo using imaging modalities and efficacy studies. To conclude, in order to exploit the advantages of actively targeted NCs, several criteria and considerations should be systematically addressed. Furthermore, additional tools, which will enable controlling the ligand density on NCs and will allow systematic study of the targeted NC–receptor interaction are lacking and must be developed.While developing actively targeted NCs, various design and biological considerations must be taken along with building a suitable experimental system to evaluate the efficacy of these systems Fig. . In the 78 that described temperature-responsive liposomes for enhanced local drug release by hyperthermia. Since then, extensive studies in this arena have explored many new materials and stimuli amenable to the development of targeted and stimuli-responsive NCs in cancer therapy. Making NCs responsive to the tumor microenvironment enables spatio-temporal control of therapeutic release, offering a promising strategy to overcome the issue of premature release from NCs in systemic circulation. Designing stimuli-responsive NCs involves the use of biocompatible materials that can undergo hydrolytic cleavage, protonation, or a conformational change in response to an intrinsic or extrinsic stimulus. This approach can therefore be simple or highly complex, depending on the choice of the stimulus and the response/sensitivity of the chosen materials. Although a wide range of stimuli and a diverse library of responsive materials enable great flexibility in designing stimuli-responsive NCs, their bench-to-bed side translation is not simple. One major obstacle is the complex chemistry involved in most of these systems, which complicates their pharmaceutical development and scalability. Variations in intrinsic stimuli like pH, enzymes, and reducing agents between preclinical and clinical models also pose a challenge  and the blood–tumor barrier84. With multiple sessions of focused ultrasound they demonstrated improved therapeutic effect of liposomal doxorubicin in rat glioma model.In addition to eliciting spatiotemporally controlled drug release from NCs, external stimuli, especially magnetic field, and ultrasound can also be used for tumor localization of NCs. This can be achieved by two different approaches. In one approach, the external stimuli may directly guide the NCs to the tumor site. For example, Foy et al. demonstrated that a short (1 h) exposure of a murine tumor to magnetic field resulted in enhanced accumulation of systemically administered magnetic nanoparticles (MNP) in the tumor85. Compared to non-targeted or single targeted nanoparticles, the combination of active targeting and magnetic targeting strategies resulted in increased nanoparticle accumulation in the tumor tissue and improved therapeutic efficacy. Similar findings have been reported by Cui et al. who developed a dual-targeting strategy based on PLGA nanoparticles combining the magnetic field guidance and TfR binding42.In another interesting approach, external stimuli mediated tumor targeting can be synergistically combined with active targeting, resulting in enhanced accumulation of NCs in the tumor tissue and increased therapeutic efficacy. For example, Schleich et al. developed RGD grafted PLGA-based nanoparticles loaded with paclitaxel and superparamagnetic iron oxides for dual targeting of tumor as a result of magnetic targeting and active targetingAlthough promising, the approach of combining external stimuli mediated targeting and active targeting may involve complex design, which may impact translatability. Therefore, careful consideration must be given to the design criteria of NCs aimed toward such dual targeting approaches. Additional challenges include limited tissue penetration of the external stimuli and compliance with providing the stimuli.86. These wafers are placed along the surface of the resection cavity during surgery and can sustain release of drug for 3 weeks. However, poor penetration of the drug through the interstitial matrix and the invasiveness of the tumor cells that spread around the brain limit the efficacy of these wafers. To resolve this issue, Sawyer et al. combined polymeric controlled release with convection-enhanced delivery (CED) and developed camptothecin (CPT)-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles that are guided via CED to a stereotactically defined location in the brain. This allows simultaneous control of location, diffusion, and duration of drug release86.Despite their immense potential, multiple physiological barriers must be overcome by passively and actively targeted NCs to achieve improved efficacy in clinic. Since bypassing systemic administration can help overcome such barriers, local delivery of the therapeutics directly into the diseased compartment is an attractive strategy. Certain compartments in the body including lung, bladder, brain, peritoneum, and eye can be considered unique as they can be accessed locally for the administration of therapeutics Fig. . Local d87. Actively targeted hyaluronan (HA)-coated lipid nanoparticles (HA-LNPs) were administered locally to the brain in a U87MG orthotopic glioblastoma mouse model. These HA-LNPs encapsulating siRNA against Pololike kinase 1 (a G2/M transition regulator) demonstrated 80% gene knockdown in vivo and prolonged survival (≥60%) compared to control mice. In another example, Liu et al. developed implantable nanofibers for localized, near infrared (NIR) triggered delivery of doxorubicin42. These studies demonstrate the strong advantage of combining local delivery with other approaches, thus bypassing formerly intractable physiological barriers while augmenting therapeutic internalization and release within the target cells.Local delivery of therapeutics in tumors can be further improved by developing hybrid approaches combining local drug delivery with other approaches, including active-targeting strategy and stimulus triggered drug release. Recently, Cohen et al. utilized a hybrid approach combining active-targeting strategy with local drug delivery88. Use of NCs in such cases can further improve efficacy due to prolonged local residence time of the therapeutics and their controlled release. As an example, tumor bed implantation of paclitaxel eluting polymeric films composed of poly(glycerol monostearate-co-caprolactone), following complete surgical resection showed superior efficacy compared with intravenous paclitaxel in preventing loco-regional recurrence, improving overall survival in murine recurrent sarcoma and non-small cell lung cancer models90.Even though this approach may be limited to certain specific cancers, it has immense potential, especially for localized chemotherapy of primary tumors that have not metastasized and when surgical resection is contraindicated. Also, for debulking surgeries that require either adjuvant or neoadjuvant chemotherapy to minimize loco-regional recurrence, localized delivery of chemotherapeutics may result in better therapeutic outcomes with reduced toxicity compared to systemic chemotherapy. For example, intraperitoneal chemotherapy before or after debulking surgery in ovarian cancer has shown therapeutic outcomes superior to systemic therapyClinical success of localized therapeutic delivery approaches in cancer treatment requires addressing multiple challenges including physiological and technological barriers that are specific to the type of cancer, as indicated in Fig. 91. The FDA and later on the European Medicines Agency (EMA) adopted regulations dictating minimal standards for conducting pre-clinical laboratory studies, aimed at the commercialization of therapeutics regulated by these regulatory authorities. We argue that adopting GLP in academic pre-clinical studies is crucial to promote academia-industry collaborations, which will help in the clinical translation of academically discovered therapeutics in general, and in particular NCs. However, it is also important to realize that not all academic pre-clinical studies necessarily require GLP. For example, incorporating GLP is not crucial and may be too labor intensive for preclinical studies that aim to establish proof of concept for a new biological mechanism or technology. In some cases, it might even slow down the translation. On the other hand, GLP is truly critical when demonstrating the promise of the technology in comprehensive functional pre-clinical models with the aim of translation. Since GLP adoption can significantly increase the overall costs of a preclinical study, careful consideration of the aims of the study may help determine whether GLP is required or not. We also argue that funding organizations should allocate funds specifically for GLP, especially for proposals that have already demonstrated proof of concept and are aimed at translation.In addition to the challenges discussed above, obstacles related to commercialization and approval of NCs by the regulatory authorities are to date the most prominent hurdles that need to be overcome to bridge the bench-bed gap. One such obstacle is the rarity of good laboratory practices (GLP) conditions and questions regarding the validity and reproducibility of scientific results in the academic setting is a barrier to their collaboration with the pharmaceutical industry. GLP guidelines were established in response to the discovery of problems with quality and data integrity in several toxicology studies in the 1970s92. This initiative defined five major criteria to be addressed: physio-chemical characterization, preclinical models, risk characterization, risk assessment, and risk communication. A direct example of the fruits of this initiative is the establishment of the Nanotechnology Characterization Lab (NCL), which performs thorough evaluations of nanoparticles received from academia, government, and industry. A standardized analytical cascade is employed to test physiochemical properties, pre-clinical toxicology, pharmacology, and efficacy both in vitro and in vivo  failed to achieve the primary end-points in two different phase 2 clinical trials for treating non-small cell lung cancer and advanced cervical and head and neck cancer, respectively. We believe that this failure stresses the need for a paradigm shift in designing clinical trials with NCs for cancer therapy. Patient pre-selection based on the extent of EPR, the presence of target receptor and tumor heterogeneity, the ability of the actively targeted NC to bind to the target receptor and the need for companion diagnostics are crucial for achieving better therapeutic outcomes. A pioneering clinical study in that direction was conducted recently by Merrimack Pharmaceuticals to determine if tumor accumulation of ferumoxytol (FMX) iron nanoparticles, as determined by quantitative MRI, may predict response to nanoliposomal irinotecan So should one uses active cellular targeting approach? Well it is all depends on the pathological location of the tumor, it’s heterogeneity, and it’s biology . Passive targeting approach has great therapeutic potential; however, it is currently limited by the heterogeneity of the EPR effect and the physiological barriers associated with it. Development of rapid quantitative EPR-imaging technologies is therefore crucial for assessing the EPR effect. Applying such imaging technologies in humans should be enthusiastically pursued, as this could be a relatively quick source of critical information for future NC design. Furthermore, such technologies could provide clinicians with companion diagnostic tools useful in pre-selection of patients who would respond to EPR-based therapies, improving therapeutic outcomes. Actively targeted NCs can offer great advantages in treating hematological malignancies; however, in solid tumors they must rely on passive targeting for tumor accumulation, and therefore suffer from the same limitations as passively targeted NCs. Cellular barriers to successful intracellular delivery of the encapsulated therapeutics and complex design are some additional factors specific to the actively targeted NCs that limit their clinical success. To overcome these challenges, we have devised criteria and considerations that must be taken into account while developing actively targeted NCs.The design of passively or actively-targeted NCs should also consider the deposition of a protein corona in physiological environment, and its consequences on biodistribution, targeting, release of therapeutics, therapeutic efficacy, and toxicity. New strategies to develop NCs with controllable/predictable biological identity are therefore necessary for accelerating the clinical translation of NCs. For the development of stimuli-responsive NCs to prevent premature release, other design challenges, such as the sensitivity of the NCs to the stimuli, scalability, and toxicity of the NCs, should also be carefully.Several approaches have been suggested for bypassing the EPR effect, including strategies that utilize exogenous cell-mediated delivery of NCs to deliver therapeutics or immune-modulating payloads in tumors. Utilizing such strategies might provide real breakthroughs in the treatment of low-EPR solid tumors, hematological tumors, and metastatic lesions. Local delivery of therapeutics using NCs, implants, hydrogels, etc., can bypass physiological barriers associated with EPR, acting as an attractive strategy in the treatment of locally accessible tumors.Finally, the development of better and more predictive pre-clinical animal models and adoption of GLP and standardization guidelines in academia are necessary to bridge the bench-bed gap. Such standardizations  coupled with better understanding of tumor biology and identification of real biomarkers that can predict responders and non-responder in advance will most likely increase the success rate of translation of novel NCs into clinical practice. The Nanotechnology Regulatory Science Research Plan and similar programs established by the health authorities should be utilized to evaluate the efficacy and safety of NCs, thereby unleashing their true potential.


Estrogens (E2) exert a plethora of neuroprotective actions against aged-associated brain diseases, including Alzheimer's disease (AD). Part of these actions takes place through binding to estrogen receptors (ER) embedded in signalosomes, where numerous signaling proteins are clustered. Signalosomes are preferentially located in lipid rafts which are dynamic membrane microstructures characterized by a peculiar lipid composition enriched in gangliosides, saturated fatty acids, cholesterol, and sphingolipids. Rapid E2 interactions with ER-related signalosomes appear to trigger intracellular signaling ultimately leading to the activation of molecular mechanisms against AD. We have previously observed that the reduction of E2 blood levels occurring during menopause induced disruption of ER-signalosomes at frontal cortical brain areas. These molecular changes may reduce neuronal protection activities, as similar ER signalosome derangements were observed in AD brains. The molecular impairments may be associated with changes in the lipid composition of lipid rafts observed in neurons during menopause and AD. These evidences indicate that the changes in lipid raft structure during aging may be at the basis of alterations in the activity of ER and other neuroprotective proteins integrated in these membrane microstructures. Moreover, E2 is a homeostatic modulator of lipid rafts. Recent work has pointed to this relevant aspect of E2 activity to preserve brain integrity, through mechanisms affecting lipid uptake and local biosynthesis in the brain. Some evidences have demonstrated that estrogens and the docosahexaenoic acid (DHA) exert synergistic effects to stabilize brain lipid matrix. DHA is essential to enhance molecular fluidity at the plasma membrane, promoting functional macromolecular interactions in signaling platforms. In support of this, DHA detriment in neuronal lipid rafts has been associated with the most common age-associated neuropathologies, namely AD and Parkinson disease. Altogether, these findings indicate that E2 may participate in brain preservation through a dual membrane-related mechanism. On the one hand, E2 interacting with ER related signalosomes may protect against neurotoxic insults. On the other hand, E2 may exert lipostatic actions to preserve lipid balance in neuronal membrane microdomains. The different aspects of the emerging multifunctional role of estrogens in membrane-related signalosomes will be discussed in this review. Sex steroids produced by peripheral glands such as dihydrotestosterone (DHT), testosterone (T), estradiol (E2), progesterone (PROG), and corticosterone (CORT) are traditionally known to play crucial roles in sexually dimorphic circuits located in the hypothalamus and other brain areas controlling reproductive behaviors, as well as brain masculinization, brain connectivity, and neuroplasticity  exert a plethora of neuroprotective actions against aged-associated brain diseases, including AD. Part of these actions takes place through binding to estrogen receptors (ER) embedded in signalosomes, where numerous signaling proteins are clustered. Signalosomes are preferentially located in lipid rafts which are dynamic membrane microstructures characterized by a peculiar lipid composition enriched in gangliosides, saturated fatty acids, cholesterol, and sphingolipids. Rapid E2 interactions with ER-related signalosomes appear to trigger intracellular signaling ultimately leading to the activation of molecular mechanisms against AD.We have previously observed that the reduction of E2 blood levels occurring during menopause induced disruption of ER-signalosomes at frontal cortical brain areas. These molecular changes may reduce neuronal protection activities, as similar ER signalosome derangements were observed in AD brains. The molecular impairments may be associated with changes in the lipid composition of lipid rafts observed in neurons during menopause and AD. These observations indicate that the changes in lipid raft structure during aging may be at the basis of alterations in the activity of ER and other neuroprotective proteins integrated in these membrane microstructures.Moreover, E2 is a homeostatic modulator of lipid rafts. Recent work has pointed to this relevant aspect of E2 activity to preserve brain integrity, through mechanisms affecting lipid uptake and local biosynthesis in the brain. Some findings have demonstrated that estrogens and the docosahexaenoic acid (DHA) exert synergistic effects to stabilize brain lipid matrix. DHA is essential to enhance molecular fluidity at the plasma membrane, promoting functional macromolecular interactions in signaling platforms. In support of this, DHA detriment in neuronal lipid rafts has been associated with the most common age-associated neuropathologies, namely AD and Parkinson disease.Altogether, these findings indicate that E2 may participate in brain preservation through a dual membrane-related mechanism. On the one hand, E2 interacting with ER related signalosomes may protect against neurotoxic insults. On the other hand, E2 may exert lipostatic actions to preserve lipid balance in neuronal membrane microdomains. The different aspects of the emerging multifunctional role of estrogens in membrane-related signalosomes will be discussed in this review.Perimenopause and menopause are important periods of woman's life where significant physiological changes occur. Perimenopause is a period in women whose age range is between 40–58 years, followed by a transition period of 1–5 years that culminates with ~12 months of amenorrhoea , the majority of symptoms of these periods are neurological in nature  regulation. A big body of data has reported a number of mechanisms by which estrogens  protect against different neuroinflammatory and neurodegenerative disorders. Thus, E2 has been demonstrated to exert beneficial actions against a wide range of diseases: AD, PD, ischemia, schizophrenia, multiple sclerosis, hypertensive encephalopathy, spinal cord injury, traumatic brain injury, and retinal degeneration  classically represents a small fraction of the total amount of receptors within the cell. ER molecule lacks either transmembrane domains, hydrophobic residues or other structural modifications to be inserted into the plasma membrane  that are activated upon specific extracellular stimulation, thereby inducing distinct cell responses. In this scenario, numerous lipid raft-associated proteins have been shown to be involved in nervous system functioning. The list includes GPI-anchored receptors; G protein-coupled receptors ; glutamate receptors ; neurotrophin receptors ; Src family receptors ; cell adhesion molecules ; and proteins associated with myelin glycosynapse . The association of this plethora of proteins in signalosomes has been shown to modulate synapsis, neuronal plasticity, cell-cell communication, myelin organization and stability, autophagy, neuronal survival, and neurodegeneration  gating modulation. This channel appears associated with ERα in neuronal lipid rafts from a wide variety of brain regions, including septum, hippocampus, and cortex from, both, murine and human origins , alterations in the proportion of polyunsaturated fatty acids (PUFA), increase in saturate/unsaturated ratio, and decrease in ganglioside or cholesterol levels that induce cell aging and death  is a PUFA highly abundant in the human brain , where it is a major component of cell membrane phospholipids. Paradoxically, the brain has very poor capacity to produce DHA , and its uptake into the plasma membrane  and acetyl-coenzyme A acetyltransferase (ACAT) genes  have been characterized. In this sense, ApoE4 is the greatest genetic risk factor to develop sporadic AD, and this risk is greater in women than men  may be considered a logical intervention. Nevertheless, ERT approaches are still a matter of controversy, because of the increased rates of stroke, coronary heart disease, and breast cancer reported in menopausal women following this preventive therapy  administrated to women at 50–63 years old might protect against cognitive decline occurring during normal aging (Henderson, An alternative intervention that appears to alleviate the impact of metabolic changes during menopausal periods is based on nutritional supplementation with vegetable phytoestrogens (Villa et al., Furthermore, emerging data discussed in this review have demonstrated a synergistic effect of, both, E2 and essential fatty acids such as DHA in the maintenance of neural homeostasis and preservation against neurodegeneration (Fabelo et al., In conclusion, we believe that nutritional-hormonal interventions may be a potential therapeutic strategy to alleviate some of the symptoms associated with, both, menopause and age-related brain degeneration, particularly at the stage of earliest recognizable symptoms. These strategies may act as compensatory mechanisms to palliate the loss of essential cell membrane lipids that ultimately may lead to neuronal dysfunction and brain detriment. Notwithstanding these encouraging results, further studies will be needed to tackle optimal strategies to mitigate menopausal effects in the nervous system.RM is the main contributor to this work; RM and MD have written the main body of the text, and provided the content of the different sections of this review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glioblastoma is known to be one of the most lethal and untreatable human tumors. Surgery and radiotherapy in combination with classical alkylating agents such as temozolomide offer little hope to escape a poor prognosis. For these reasons, enormous efforts are currently devoted to refine in vivo and in vitro models with the specific goal of finding new molecular aberrant pathways, suitable to be targeted by a variety of therapeutic approaches, including novel pharmaceutical formulations and immunotherapy strategies. In this review, we will first discuss current molecular classification based on genomic and transcriptomic criteria. Also, the state of the art in current clinical practice for glioblastoma therapy in the light of the recent molecular classification, together with ongoing phases II and III clinical trials, will be described. Finally, new pharmaceutical formulations such as nanoparticles and viral vectors, together with new strategies entailing the use of monoclonal antibodies, vaccines and immunotherapy agents, such as checkpoint inhibitors, will also be discussed. Gliomas are the most common primary central nervous system (CNS) tumors and, among these, half of all new diagnosis is represented by glioblastoma (GBM), the most malignant type of brain cancer with a poor prognosis and a median patient survival of approximately 18 months [Recently, the new World Health Organization (WHO) Classification of Tumors of the CNS  includedIn addition, as a result of genomic profiling and the Cancer Genome Atlas Project , more thMolecular alterations of gene expression patterns have been assessed between primary and secondary gliomas. Genetic alterations typical for primary GBM are epidermal growth factor receptor (EGFR) gene mutation and amplification, phosphate and tensin homologue (PTEN) mutations and chromosome 10q loss. In secondary GBM, isocitrate dehydrogenase 1 (IDH1) mutations, p53 mutations, and chromosome 19q loss are frequently found . In addiDNA-methylation analysis is a useful tool to distinguish glioblastoma subgroups associated with specific epigenetic and genetic features. On this basis, four subgroups of adult glioblastoma have been identified, including an IDH mutant, and three IDH wild-type glioblastoma subgroups; the IDH mutant subtype has methylated O6-methylguanine methyltransferase (MGMT) promoter, displays a number of chromosomic aberrations and other alterations such as MYC activation, upregulation of the receptor tyrosine kinase (RTK)/Ras/PI3K pathway and mutations of genes encoding inhibitors of the G1/S cell-cycle checkpoint, including the Rb pathway ,11.The IDH wild-type glioblastoma subgroups have different DNA-methylation profiles and a fewer copy-number aberations. The so called “receptor tyrosine kinase I” (RTK I) glioblastomas are characterized by platelet-derived growth factor receptor A (PDGFRA) amplification and are mainly diagnosed in young subjects.The “receptor tyrosine kinase II” (RTK II) and the “mesenchymal” subtypes are generally diagnosed in older patients (from 50 years of age) and the mesenchymal glioblastoma shows a mesenchymal gene-expression profile and is related to radioresistance and shorter survival .Alterations of miRNA levels have been found in almost all cancer biology processes, including cell proliferation, migration, angiogenesis, and chemoresistance. Alterations of GBM miRNAs have been reported and a very recent study identified a panel of miRNAs that are likely to be dysregulated by genomic deletions and amplifications. In addition, the authors found that a miRNA acting as a tumor suppressor, miR-4484, is deleted in GBM, thereby leading to deregulation of a panel of genes involved in various cancer-related processes .In the future, the need to distinguish between neoplastic cells and surrounding cells belonging to tumor niche, in the aim of finding molecular targets for glioma therapy, will represent a real challenge. Recently, two interesting papers have addressed this issue using single-cell analysis coupled to Next-Generation Sequencing (NGS) techniques. In the first study, single cell analysis of cells from the tumor core and surrounding tissue found a wide cellular variation in the tumor genome and transcriptome. Conversely, infiltrating GBM cells appeared to share a consistent gene signature between patients, suggesting the existence of common features underlying the infiltration mechanisms .In another report  single cCurrent standard therapy for GBM includes surgical resection, followed by radiation and co-administration of temozolomide (TMZ), an oral alkylating agent. Extensive surgical resection, though representing the most effective way to increase survival of GBM patients, is hardly feasible depending on tumor localization and infiltration, particularly when highly specialized brain areas are involved, such as those involved in the control of speech, motor function and senses. However, the highly infiltrative behavior of GBM makes surgery nearly ineffective, since tumor cells and glioblastoma stem cells (GSC) colonize the surrounding brain tissue causing relapses located even at distant brain sites . NeverthIn addition to these techniques, the use of Gamma Knife radiosurgery (GKRS) has provided advantages especially for recurrent glioblastoma (rGBM). GKRS is a type of stereotactic radiosurgery capable of delivering a high dose of radiation to a tumor, sparing healthy surrounding tissue and is particularly used in rGBM. GKRS does not require open resection and allows minimization of radiation-induced necrosis to surrounding brain tissue . The maiHyperthermia is another strategy adopted in the treatment of GBM patients. Hyperthermia brings tumor tissue temperature to a range of 41–46 °C, thereby inducing physiological changes in tumor cells, such as protein misfolding, aggregation, alteration of signal transduction pathways, eventually leading to apoptosis . The effHowever, despite these technologies, differentiating between normal brain and tumor tissue continues to be a major challenge and, even with advances in surgical resection, the prognosis for GBM patients remains poor.On the pharmacological therapy side, great efforts have been made to develop new pharmacological tools leading to a series of clinical trials recently completed or currently ongoing .The classical clinical practice includes TMZ treatment for GBM patients. TMZ is a derivative of the alkylating agent dacarbazine and is active against human cancers such as melanomas and astrocytomas by inducing cell cycle arrest at G2/M phase and eventually leading to apoptosis ,22,23. PTo overcome this resistance mechanism, the combination of capecitabine and TMZ was designed. Capecitabine is a prodrug of the pyrimidine analog 5-FU (5-fluorouracil), which is enzymatically converted to 5-FU and leads to inhibition of MGMT repair activity, probably through depletion of MGMT protein and mRNA ,28,29,30Nanoparticles (Nps) are structures in the nanometer size range having different structures and properties and, in general, are widely studied for biomedical applications . In GBM Lipid carriers are bilayered vesicles composed of phospholipid membranes (liposomes). Liposome phospholipids are mainly represented by biocompatible fatty acids, such as phosphatidylcholine and ethanolamine, and display on their surface both hydrophilic and hydrophobic regions . These vInorganic Nps can be utilized for diagnostic and therapeutic purposes. Magnetic Nps (MNps) are the most common Nps used in biomedical applications due to their high biocompatibility; injectable super paramagnetic iron oxide Nps (SPION) can be also used in MRI with lower toxicity effects and higher sensitivity compared to conventional contrast agents . In addiAn interesting application is the use of Nps in combination with siRNAs; it is known that stimulation of the thyrosin kinase receptor c-Met induces proliferation of glioma cells and resistance to chemotherapy; intravenous administration of polyethylene glicol (PEG) Nps loaded with c-Met siRNA reduced c-Met expression and powerfully inhibited cell proliferation and resistance to chemotherapeutic agents in GBM cells .Glioblastoma stem cells (GSCs) are currently held responsible for GBM infiltration in the brain tissue, proliferation and resistance to chemotherapy . For theIt was once thought that the central nervous system was devoid of normal immunologic function and lymphatic vessels . Indeed,Furthermore, under physiologic conditions, the brain hosts several populations of immune cells such as microglia, which stem from hematopoietic cells and colonize CNS during embryonic development. Microglial cells migrate to inflammatory sites in the CNS, activate and behave like phagocytes or antigen presenting cells, secreting cytokines and chemokines .This picture is different in GBM patients, who display a slight but detectable immune suppression condition, compared to the general population, and low adaptive immune responses. In addition, the tumor microenvironment is rich in immunosuppressive factors secreted by the tumor, like transforming growth factor beta (TGF-β) and vascular endothelial growth factor (VEGF) that suppresses cytotoxic T cell activity .In this background, different approaches are under investigation: in targeted therapies, immune cells or antibodies directed against tumor antigens are given to patients and this approach does not require activation of patients’ immune system. Over recent years several molecular targets have been identified and tested in GBM therapy, with controversial results .In GBM the so-called EGFRvIII mutation is frequently found; this mutation arises from a deletion of 267 amino acids of the extracellular domain leading to the expression of mutants with a unique extracellular domain . This muSome monoclonal antibodies (mAbs) binding to EGFRvIII have been developed but, despite the relevant results of antibody-based therapy in the treatment of other cancer types such as renal carcinoma, breast cancer, melanoma, and hematologic cancers, these results have not been equally successful in GBM . Among tIndeed, the first mAb tried in the therapy of GBM patients was bevacizumab. Bevacizumab is a humanized monoclonal antibody that binds to the different forms of the vascular endothelial growth factor (VEGF). Bevacizumab is currently used in the therapy of a variety of tumor types and is also in trial for GBM therapy. However, in recent, randomized, double-blind, placebo-controlled studies designed to evaluate first-line use of bevacizumab together with the standard therapy, chemoradiation and TMZ, no increase of median overall survival was reported. It should be also noted that patients were stratified by MGMT promoter methylation and other gene mutations but no group showed benefits from bevacizumab treatment  with, inNew insights could still come from ongoing clinical trials but at the moment classical antibody-based approaches are making time.A promising possibility is represented by the use of mAbs linked to cytotoxic molecules that specifically target cancer cells, thereby delivering drugs or toxins; these mAbs are named antibody drug–conjugate (ADC) and are under investigation for the use in GBM patients . ImmunotAnother interesting target is the αvβ3 integrin receptor, which is highly expressed in glioblastoma and can be targeted by mAbs or by integrin receptor ligands conjugated to cytotoxic drugs; preclinical studies are giving promising results .Immunotherapy offers a different approach from chemotherapy, targeted therapy, radiation and surgery, raising new hopes, particularly for immune checkpoints inhibitors. T lymphocytes can recognize antigens expressed by cancer cells but, conversely, immune checkpoints, particularly programmed cell death (PD)-1 receptor and its ligand (PD-L1), can suppress the activity of T lymphocytes by inducing apoptosis in activated immune cells ,47. The The growing interest in immune checkpoint inhibitors and the encouraging results in the treatment of these cancer types have driven in recent years the attention of researchers towards the use of anti PD-1 and PD-L1 mAb in GBM therapy. Preclinical studies in mice have given promising results and a number of clinical studies involving pembrolizumab and nivolumab alone or in combination with other agents  are starting or still ongoing and results will be available in a few years.Also the very new therapies involving the use of genetically modified T cells expressing chimeric antigen receptors (CARs) may represent a new offensive strategy in the GBM siege. These T cells are modified to specifically recognize GBM antigens such as IL13Rα2, HER2, EphA2, and EGFRvIII, the first antigens to be tried in this type of immunotherapy ,51, and Oncolytic viruses (OV) specifically target cancer cells; OV have been designed to take advantage of tumor-specific mutations, or signaling pathways that are constitutively activated in tumors, and are selected to enter tumor cells overexpressing tumor antigens . The infSeveral OV are under investigation for high-grade glioma therapy and, among these, two types of modified Herpes simplex virus 1 (HSV1) and adenovirus (AdV) are in clinical trial. Indeed, a variety of oncolytic HSV mutants such as R3616, HSV-1716, hrR3, G207, and G47Δ have been designed for targeting glioblastoma. All these mutants display deleted or mutated viral genes, thus reducing neurotoxicity while not affecting infection of dividing cells, particularly those with activated Ras pathway signaling . In G207AdV is a non-enveloped virus with a double-stranded, linear DNA genome. There are multiple engineered versions under investigation, including DNX-2401 and ADV-TK that are in clinical trial. DNX-2401 mutation allows for replication only in cells bearing Rb tumor suppressor deletion . The tumADV-TK is an adenoviral vector engineered to express the Herpes thymidine kinase gene followed by administration of an anti-herpetic prodrug (ganciclovir—GCV). A randomized phase II clinical trial demonstrated progression-free survival and overall survival benefit associated with ADV-TK gene therapy, which was also shown to be safe .Another interesting therapeutic strategy in clinical trial is represented by the use of Toca 511 and Toca FC . These aThe EGFRvIII mutation occurs in roughly 20–30% of all glioblastomas and its potential immunogenicity has raised substantial interest in the neuro-oncology field; the studies aimed at finding new targets for glioblastoma therapy resulted in the development of rindopepimut, a peptide vaccine containing the amino acid sequence found in the EGFRvIII deletion mutation conjugated to keyhole limpet hemocyanin. In four separate glioblastoma trials, ACTIVATE, ACT II, ACT III,  rindopepOther glioblastoma vaccines are under investigation in single-arm and randomized Phase II trials and results will be available in a short time.Evidence coming from preclinical studies and clinical trials indicate that strategies to counteract GBM aggressiveness are multiplying and that knowledge of GBM mutations and genetic profiling can highlight possible molecular targets for therapies. It is also clear that traditional therapies such as surgery, chemotherapy and radiation still remain the first line approaches to GBM and hopes to improve life quality and overall life expectancy of GBM patients mainly lie, at the moment, in the new immunotherapy strategies. In the coming years a number of clinical trials will give fundamental insights about these therapeutic approaches and, contemporarily, basic research must pursue the objective of discovering new mechanisms and new targets to help and direct clinical research.


Likewise, men experiencing problems with emotion regulation, a multidimensional construct reflecting difficulties in effectively experiencing and responding to emotional states, are also at risk of IPV perpetration. In the present research, we tested the hypothesis that the link between discrepancy stress and IPV perpetration is mediated via difficulties in emotion regulation. Three hundred fifty-seven men completed online surveys assessing their experience of discrepancy stress, emotion-regulation difficulties, and history of IPV perpetration. Results indicated that discrepancy-stressed men's use of physical IPV was fully mediated by emotion-regulation difficulties. In addition, emotion-regulation difficulties partially mediated the association between discrepancy stress and sexual IPV. Findings are discussed in terms of the potential utility of emotion-focused interventions for modifying men's experience and expression of discrepancy stress and reducing perpetration of IPV.Research suggests that masculine socialization processes contribute to the perpetration of intimate partner violence (IPV) by men. Although this research has traditionally focused on men who strongly adhere to traditional gender norms, men who negatively evaluate themselves as falling short of these norms (a construct termed  Intimate partner violence (IPV) constitutes a significant and preventable public health problem with consequences ranging from loss of productivity, negative physical and mental health outcomes, and even death. Indeed, in a recent large-scale epidemiological study, IPV resulted in an estimated 2,340 deaths per year—accounting for 14% of all homicides . Importadiscrepancy stress  of emotion-regulation difficulties that underlie men's IPV perpetration is essential to the development of effective, tailored interventions. Contemporary models of emotion regulation include acceptance, understanding, and awareness of emotions as well as the ability to inhibit impulsive behaviors and flexibly modulate emotions in the service of superordinate goals e.g., . DifficuEmotion-regulation difficulties have been identified as a prominent component operating in both men and women's perpetration of IPV ; howeverThe pressure to over-control emotional experiences may be particularly salient for men experiencing DS, who are likely prone to gender-threatening thoughts. These thoughts, in turn, may produce “vulnerable” emotions , which would exacerbate DS given the incongruence between these emotions and masculine norms. Consequently, discrepancy-stressed men may be more likely to avoid or suppress these vulnerable emotions to express emotions more congruent with masculine norms , despite the arousing effects of emotional suppression . TherefoEvidence suggests that men who negatively evaluate themselves as falling short of masculine norms may be at increased risk of IPV perpetration . ImportaThe current study constitutes a secondary analysis of survey data collected by Reidy and colleagues in 2012 with the aim of testing the hypothesis that emotion-regulation difficulties mediate the relationship between DS and IPV. Baseline evidence for a significant relationship between DS and IPV by men was first established in this sample and has been reported elsewhere . In the Participants were 405 U.S. men recruited via Amazon's Mechanical Turk (MTurk) website who indicated they “had been in an intimate relationship within the prior 12 months.” MTurk permits the collection of national data from individuals via an online method that proffers valid and reliable data with more diversity in samples than traditional convenience samples . IndividParticipants responded to a series of questions about age, ethnicity, marital status, relationship history, self-identified sexual orientation, and level of education.Respondents answered 5-point scale questions (1 = “strongly agree” to 7 = “strongly disagree”) pertaining to the experience of DS . This scale  has beenThe CTS-2  instructClarity subscale reflect the extent to which individuals know the emotions they are experiencing . Items on the Nonacceptance subscale assess lack of acceptance of emotions . The Goals subscale reflects an inability to engage in goal-directed behavior when distressed , while the Impulse subscale reflects impulse control difficulties when experiencing negative affect . An Awareness subscale contains items measuring difficulties attending to ongoing emotional experiences . Last, the Strategies subscale assesses limited access to strategies for effective regulation of emotions . The DERS has demonstrated adequate convergent validity with established measures of emotion-regulation difficulties and emotional avoidance and has been demonstrated to be predictive of IPV in an undergraduate sample . The final analytic sample comprised 357 men with a mean age of 28.1 (SD = 6.9). The sample was largely consistent with the general U.S. population in terms of ethnicity  and income , but slightly more educated  compared with the general population of men  on the demographics questionnaire were excluded from analyses. In addition, we removed seven respondents who were more than three standard deviations from the mean time of completion on the survey  on all six DERS subscales. Results are provided in Path analyses specifying Physical Assault and Sexual Coercion as outcome variables were tested in separate multiple mediator models to estimate indirect effects between DS and IPV perpetration via difficulties in emotion-regulation domains. All variables were standardized prior to analyses to permit meaningful comparison across coefficients. Bootstrap estimates were calculated to examine indirect effects, as this method does not require assumption of normality in the sampling distribution . All indBootstrap estimates of the direct effect coefficients of DS on IPV perpetration controlling for all DERS subscales were also calculated see . For PhyThe purpose of the current study was to build on extant literature regarding risk factors for men's perpetration of violence against their intimate partner . SpecifiFirst, we found a significant relationship between DS and emotion-regulation difficulties. Extant theory and data suggest that the anxiety-producing effects of perceiving oneself as falling short of precarious masculine standards render men vulnerable to emotion-regulation difficulties by encouraging the overreliance on avoidant emotion-regulation skills and impeding the development of more flexible skills for experiencing and expressing emotions directly e.g., . HoweverThese findings may be associated with the norm violating nature of DS itself. In other words, men who are distressed by self-perceived deficiencies in their masculinity must contend with vulnerable emotions. These emotions may, in turn, be perceived as further violations of masculinity given prevailing norms dictating toughness and restricted emotional expression e.g., . The expunique effect of each domain of emotion-regulation difficulties on IPV. Specifically, we found that the Clarity and Impulse subscales significantly predicted the frequency at which men in our sample utilized physical IPV, even when controlling for their competencies in other emotion-regulation domains. These results suggest that men who struggle to accurately interpret and label emotional experiences and those with problems inhibiting impulsive behavior when distressed may be at particular risk of physical IPV perpetration. Difficulties inhibiting impulsive behavior when distressed also predicted the frequency of sexual IPV perpetration. These results suggest that difficulties in any one of those significant emotion-regulation domains may be sufficient, in isolation, to engender risk of IPV.Second, consistent with previous studies and emotion-regulation models of aggression, results of the current study revealed significant associations between emotion-regulation difficulties and IPV perpetration . ImportaIndeed, results of the current study were consistent with full mediation for physical assault, such that the relationship between DS and physical IPV was fully explained by discrepancy-stressed men's emotion regulation difficulties. However, not all emotion-regulation domains contributed equally to this effect. Our findings indicate that a specific subset of emotion-regulation difficulties drove discrepancy-stressed men's risk of engagement in physical IPV against their intimate partner. Notably, in the context of DS, difficulty identifying one's emotional experience with clarity (Clarity), inhibiting automatic responses when distressed (Impulse), and believing that few strategies for coping with strong negative emotion are available (Strategies) appear to underlie risk of physical IPV perpetration. The emergence of significant indirect effects of discrepancy stress on physical IPV perpetration via these specific difficulties in emotion-regulation integrates, extends, and clarifies theory regarding the etiology of men's physical IPV. In contrast to physical IPV, discrepancy stress accounted for the men's sexual IPV above and beyond the effects of emotion regulation. Although, sexual IPV was partially explained by men's difficulties with emotional clarity and impulsivity, results of the present study indicate that discrepancy-stressed men may continue to be at risk of perpetration of sexual IPV even in the absence of emotion-regulation difficulties.The finding that emotion-regulation difficulties fully mediated the relationship between DS and physical IPV but not sexual IPV is an important and novel contribution of the present study. This finding provides preliminary evidence that physical and sexual IPV may differ in important ways, particularly as they relate to gender-socialization processes. Specifically, these findings suggest that sexual IPV is more directly related to the performance of masculinity than physical IPV and therefore, less affected by an individual's emotion-regulation skills. Abundant evidence supports a relationship between hypermasculinity and sexually aggressive behavior e.g., . As suchNotably, regarding the indirect effect of DS on physical and sexual IPV perpetration via difficulties engaging in goal-directed behavior when distressed , mediation results were in the opposite direction of other significant emotion-regulation difficulties. In other words, discrepancy-stressed men were more likely to perpetrate IPV when goal-oriented. Because aggression is seen as a highly salient method of demonstrating manhood , this paSeveral limitations of this study warrant comment. First, given the cross-sectional design of this study, causal determinations regarding interrelations among assessed variables may not be made. More research is needed to fully evaluate the theoretical proposition that masculine socialization processes in general, and DS in particular, temporally precede emotion-regulation difficulties and downstream IPV. For example, although this study provided compelling theoretical and statistical support for the supposition that engaging in IPV may provide an opportunity for men who experience DS to temporarily regain masculine status, it is also possible that engaging in IPV could, in fact, function to exacerbate DS for men who endorse proscriptive norms about violence toward women . Moreover, the present results do not address the developmental trajectory by which gender-based learning and associated stress inform one another. Although there is evidence that boys learn to perform gender “in the context of social learning that not only shapes adherence to gender norms, but also produces anxiety, fear, and other forms of restricted social and emotional functioning” see , p. 1, fSecond, utilization of self-report measures may skew the actual prevalence of IPV perpetration. Given social proscriptions against violence toward women , men in SD = 6.9), is younger than that of studies utilizing clinical samples of men court-mandated to treatment for use of IPV. As such, associations among DS, emotion-regulation difficulties, and IPV should be tested in a clinical sample of older adults, to determine whether similar patterns emerge among participants with more extensive histories of violence and behavioral health difficulties. Such investigations are necessary to ascertain whether IPV interventions focused on reducing DS, and associated emotion dysregulation may be effective in clinically relevant contexts.Last, participants in this sample were a relatively homogeneous sample of men. Although MTurk data typically proffers greater diversity than undergraduate convenience samples, the generalizability of future research would be increased by the inclusion of greater diversity across ethnicity, race, and class, particularly as masculinity is likely constructed in intersection with these variables. The mean participant age of this sample, 28.1 (Despite these limitations, the current study has significant implications for the development of targeted violence-prevention interventions. As emotion regulation may be conceptualized as a modifiable constellation of skills , the elu


Abnormalities are detectable before clinical symptoms develop, offering the potential for neuroprotective intervention in familial cases. Although promising neuroimaging biomarker candidates for diagnosis, prognosis, and disease progression have emerged, these have been from the study of necessarily select patient cohorts identified in specialized referral centers. Further multicenter research is now needed to establish their validity as therapeutic outcome measures.Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative, clinically heterogeneous syndrome pathologically overlapping with frontotemporal dementia. To date, therapeutic trials in animal models have not been able to predict treatment response in humans, and the revised ALS Functional Rating Scale, which is based on coarse disability measures, remains the gold-standard measure of disease progression. Advances in neuroimaging have enabled mapping of functional, structural, and molecular aspects of ALS pathology, and these objective measures may be uniquely sensitive to the detection of propagation of pathology The online version of this article (doi:10.1007/s13311-016-0484-9) contains supplementary material, which is available to authorized users. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the motor system and its associated neuronal networks. Pathologically it is characterized by cytoplasmic inclusions of ubiquitinated TAR DNA-binding protein 43 in degenerating upper motor neurons (UMNs) of the primary motor and frontotemporal cortices, and lower motor neurons (LMNs) of the brainstem nuclei and spinal cord anterior horns. The syndrome is heterogeneous and overlaps clinically, pathologically, and genetically with frontotemporal dementia (FTD). Progressive muscle weakness leads eventually to death, typically caused by respiratory insufficiency, with a median survival from symptom onset of only 2 to 3 years .C9orf72, SOD1, TARDBP, FUS), and asymptomatic carriers of such mutations offer a window into the earliest pathological changes [ALS is emerging as a final common pathway from multiple upstream pathological mechanisms . Approxi changes . To dateversus LMN signs is variable, with extremes of UMN involvement termed primary lateral sclerosis and those of LMN involvement, termed progressive muscular atrophy. These extremes are both associated with slower rates of progression [ALS symptoms typically begin in the distal limb or bulbar musculature, and typically spread to contiguous body regions clinically , outwardgression –9. The cgression .The revised ALS Functional Rating Scale (ALSFRS-R), which is based on coarse disability measures driven by LMN dysfunction and remote from histopathological changes, remains the gold-standard measure of disease progression , 12. Thein vivo structural and functional abnormalities observed in the brain and spinal cord [Histopathological stages of TAR DNA-binding protein 43-positive pathology based on postmortem ALS brains support concepts of prion-like connectomic spread of pathology in ALS –19. Advanal cord . This revolume of this structure. Conveniently, a number of currently available analysis tools now allow automated segmentation of various cortical and subcortical brain structures [cortical thickness and surface area measures [voxel-based morphometry (VBM) enable statistics on gray matter density maps on a whole-brain, voxel-by-voxel basis, and can provide information on regional atrophy patterns on a group level without the need for a priori assumptions [T1-weighted structural MRI results in images with good tissue contrast , and is the method of choice for the investigation of gray matter, with the added advantage that the respective sequences are readily available on clinical MRI scanners. The most basic analysis approach is to utilize the acquired images in order to outline a region-of-interest (ROI) known to be affected in a disease process and to determine the ructures . These tmeasures . Automatumptions , 24.white matter pathology [diffusion tensor imaging (DTI), which utilizes acquisition sequences sensitive to Brownian motion of water in fiber bundles [Derivatives of VBM, such as voxel-based intensitometry can quantify ALS athology , but the bundles . DTI ana bundles . Further bundles , includi bundles  or “kiss bundles . A furth bundles , 32.task-based fMRI). A more recent development called resting-state fMRI enables the investigation of the coherence of regional MRI signals in the brain at rest . Even in the absence of external stimuli any brain region will have spontaneous fluctuations in BOLD signal, allowing the exploration of the brain’s functional organization and to investigate deviations from a healthy pattern in neurological disorders. Resting-state functional connectivity research has revealed a number of networks (representing networks of brain regions which show synchronous or coherent activity) that are consistently found in healthy subjects [a priori-defined voxels or ROI are used to calculate signal correlations with other voxels in the brain. This hypothesis-driven approach depends on a consistent way to define the seed within and between patients [The most common functional MRI (fMRI) technique is based on the blood oxygen level-dependent (BOLD) response and the assumption that firing neurons cause locally increased energy demands, which result in a local increase of cerebral blood flow (CBF) and, to a lesser extent, local increases in cerebral oxygen metabolism and cerebral blood volume, in a way that creates a relative abundance of oxygenated hemoglobin in that area. Oxygenated and deoxygenated hemoglobin have different magnetic properties, which makes a change in their relative quantities detectable by MRI. Given enough repetitions of a task  performed in the scanner contrasted with rest periods, statistical methods can be used to determine the areas in the brain in which MR signal variations fluctuate in accordance with the task . Proton MRS can robustly distinguish N-acetyl aspartate , choline , and creatine . Furthermore, glutamate-related metabolites (glutamate and glutamine), as well as γ-amino butyric acid (GABA) can, depending on variables such as magnetic field strength, field homogeneity, and signal-to-noise ratio, be quantified separately or as a composite of glutamate, glutamine, GABA, and other metabolites in the brain.Magnetic resonance spectroscopy (MRS) is a means to explore the metabolite content of brain tissue PET is used to observe molecular metabolic processes in the brain using positron emitting radioisotopes (tracers) produced by a cyclotron. The tracer, injected peripherally, reaches the brain via a peripheral circulation injection. Subsequently, detectors in the PET scanner record simultaneous 180-degree pairs of gamma rays emitted as a consequence of positron-electron-annihilation, allowing 3-dimensional images that indicate the original radiotracer distribution in the brain to be reconstructed by computer analysis.18F-fluorodeoxyglucose. Similar to fMRI studies, PET brain activation studies are based on the assumption that areas of high radioactivity are related to increased blood flow or, in this case glucose metabolism, and therefore surrogates for altered regional brain activity.The most commonly used metabolic PET tracer for activation studies is Ligand PET allows visualization of neuroreceptor pools in the brain. For this purpose, tracers have been developed that are ligands for specific neuroreceptors. These may be expressed on neurons and so act as surrogates for neuronal loss, or reflect changes in neurotransmitter levels . AnotherSOD1 were also reported to have lower FA in the CSTs [The diagnostic delay in ALS (on average 1 year from symptom onset) is multifactorial and would not be completely addressed by the development of a diagnostic biomarker . The detthe CSTs , though the CSTs . Despitethe CSTs . HoweverCompared with imaging measures reflecting white matter structural integrity, results of cross-sectional VBM studies in ALS have been somewhat inconclusive. Some studies have reported gray matter differences between patients and healthy controls Significant presymptomatic structural changes were also noted in carriers of C9orf72 hexanucleotide expansions, linked to the development of ALS and FTD , 74.In addition to structural abnormalities, fMRI studies have demonstrated increased cortical activity in patients with ALS performing motor tasks , 76, as A shared signature of resting state functional connectivity changes involving the cerebellum was noted in both symptomatic sporadic patients and asymptomatic carriers of ALS-causing genetic mutations . Replicain vivo evidence for activated microglia in ALS in motor and frontal lobe regions [Preceding the wide availability of fMRI, the earliest activation PET studies also demonstrated widespread abnormalities of cerebral glucose metabolism  and chan regions , 90, wid regions  in keepi regions , and pro regions , also se regions . An emer regions , with ev regions .To date there have been no longitudinal PET studies in ALS.Investigations of metabolic tissue content by means of MRS have consistently revealed NAA , 98 and vs bulbar), which is a well-established prognostic marker [Several studies reported regional gray and white matter changes in relation to clinical and neuropsychological features, most commonly in relation to site of onset  [A third study assessed  months) . In lineTaken together these longitudinal imaging findings in ALS suggest that white-matter pathology may be significantly established in the symptomatic phase of ALS where therapeutic trials are most likely to take place, with the magnitude of change likely then greatest in those with the fastest rates of disease progression.The Alzheimer’s Disease Neuroimaging Initiative has been pivotal in developing the potential of neuroimaging outcome measures in Alzheimer’s disease therapeutic trials . SignifiClinical outcome measures in neurodegenerative disorders generally do not enable the distinction between disease-modifying and symptomatic drug effects. Symptoms might temporarily improve under the influence of a drug without a change in the actual disease activity, which could lead to an initial overestimation of a potential beneficial drug effect. Disease-modifying effects might also be missed during the trial period if they are not immediately reflected in a symptomatic response. Clinical scores are not objective and may be far removed from the underlying histopathological changes. They may be influenced by factors not directly disease-related and cannot be controlled, such as patient motivation and unwanted drug side-effects, which can result in reduced statistical power to detect therapeutic effects and consequently necessitate larger test cohorts. It has also been noted that rate of progression as defined by ALSFRS-R in patients seen soon after symptom onset is less reliable , so thatversus 567 for FA [Few longitudinal imaging studies in ALS have attempted to estimate the power of MRI in the detection of therapeutic effect. Changes of FA rates in the right superior CST were used to estimate a sample size of 263 patients per arm (placebo and treatment) to detect a hypothetical treatment effect size of 25% with 80% power . Another7 for FA .Potential safety risks associated with different brain imaging methods are an important factor that requires consideration before application in clinical trials . MRI doerobustness and reproducibility of the respective technique or analysis method must be evaluated. While in every imaging study the raw data have to pass some degree of initial quality control to exclude nondisease-related gross anatomical abnormalities and artifacts , some postprocessing algorithms require higher degrees of data quality than others in order to be able to run successfully. In research studies, data are often simply classified as not analyzable and discarded when a certain algorithm fails. However, this exclusion of data in the research setting may lead to a biased assessment of the “real-world” usefulness as a biomarker, as an algorithm that appears to be very sensitive for the detection of disease-related abnormalities, but necessitates the exclusion of some data or subjects, might ultimately require larger test cohorts if used as an endpoint in a clinical trial.Before an imaging marker can be proposed as a useful outcome measure in a clinical trial, the Another factor is the generalizability of outcomes derived from research studies that tend to be based in a single imaging center, often utilizing highly advanced image acquisition hardware and image analysis software and expertise not easily available in a clinical setting. In contrast, most advanced therapeutic trials are run in a multicenter setting, posing the problem of combining data from a variety of scanners, potentially acquired with different imaging sequences that are dictated by scanner capabilities and limited by hardware constraints. Usage of multiple scanners, as well as the fact that even on the same scanner performance is likely to change over time, necessitates harmonization of data acquisition and analysis protocols between centers and, most importantly, stringent quality control. For instance, phantom measurements can be incorporated to assess both scanner performance and the validity of postprocessing image corrections in order to reduce systematic errors in human data . This shwww.nisals.org), retrospectively combined DTI FA maps of control participants to establish correction matrices, with correction algorithms then successfully applied to the FA maps of the control and ALS patient groups [It is possible to assess systematic differences across centers when applying a harmonized acquisition protocol , and thet groups . To estat groups , and uset groups .http://imaging.ukbiobank.ac.uk), or select individuals with a high genetic risk of developing ALS in the future that have not yet developed symptoms  may help distinguish disease-related brain abnormalities from symptom-related changes and identify neuroprotective targets.An important caveat for the application of fMRI in search of potential therapeutic targets and markers of disease progression is that part of the observed differences between patients and controls, as well as changes over time revealed by imaging in ALS, might simply reflect secondary changes caused by clinical symptoms. These might be maladaptive or compensatory. Results of ongoing prospective imaging studies in large cohorts of randomly selected healthy people , only single time-point data have generally been acquired for healthy controls in the research setting.indirect measurement of neuronal activity, BOLD fMRI can only detect changes in blood flow, and does not provide information about the absolute amount of CBF. Consequently, if a drug’s effects on neurovascular coupling are unknown, a change in the BOLD signal might not only reflect its influences on neural activity, but also its potential effects on neuronal signaling to the blood vessels involved in the CBF response, or on vasculature itself. Inclusion of healthy volunteers or implementation of complementary techniques, however, can help to discern neural activity and vascular components of the BOLD signal. MR-based perfusion techniques, such as arterial spin labeling can be used to obtain quantitative CBF maps either as a single baseline image or in a time series, in order to control for global drug-related CBF changes. Arterial spin labeling fMRI is, however, technically challenging and suffers from poorer signal-to-noise ratio, and lower spatial and temporal resolution than BOLD. An alternative approach that can assist interpretation of the BOLD signal is direct quantitative measurement of neural activation using electrophysiological techniques, such as electroencephalography or magnetoencephalography [While BOLD fMRI has the advantage of being a noninvasive technique that bypasses some of the safety concerns related to the use of radioactive substances used in PET, it comes with 1 major limitation that must be considered when interpreting the BOLD signal, particularly in the context of pharmacological research. Besides only providing an lography , 134. Boin vivo evolution of cerebral pathology as a link between cellular and system dysfunction in ALS. DTI metrics are promising diagnostic biomarkers, able to identify brain white matter abnormalities closely linked with known ALS pathology, and may have a particular niche in the detection of occult UMN involvement thereby expanding the trial inclusion pool. Inconsistent findings resulting from longitudinal studies in ALS can be substantially attributed to small and clinically heterogeneous groups, and analysis methodology for some sequences, rather than insensitivity of MRI to structural and functional cerebral pathology. Ultimately, a multimodal approach combining imaging, neurophysiology, and biofluid markers may be needed to provide a signature sensitive enough to stratify and measure the effect of therapeutics on disease activity at the level of the individual patient. Meanwhile, postmortem MRI studies systematically comparing diffusion and other structural imaging metrics with histopathological markers [MRI is unique in being able to assess simultaneously brain structure and function, and provide a deeper understanding of  markers  may shed markers , and so ESM 1(PDF 1224 kb)Below is the link to the electronic supplementary material.