Acoustic metamaterials are large-scale materials with small-scale structures. These structures allow for unusual interaction with propagating sound and endow the large-scale material with exceptional acoustic properties not found in normal materials. However, their multi-scale nature means that the manufacture of these materials is not trivial, often requiring micron-scale resolution over centimetre length scales. In this review, we bring together a variety of acoustic metamaterial designs and separately discuss ways to create them using the latest trends in additive manufacturing. We highlight the advantages and disadvantages of different techniques that act as barriers towards the development of realisable acoustic metamaterials for practical audio and ultrasonic applications and speculate on potential future developments. The progression of research concerning the acoustic, vibrational and mechanical behaviour of small-scale structures is closely linked to the status of microfabrication/3D printing technologies ,2. As thMicroscale printing has only recently been demonstrated to any significant scope ,9,10, deAMMs have been studied extensively ,22,23,24A noteworthy area of AMM research is active structures, whose material properties and/or physical geometries can be changed by external stimuli. Parameters of particular interest are tuneable bulk modulus and effective density ,30,31,32This review explores the variety of small-scale AMMs currently in development, along with the 3D printing methods that show potential to manufacture them. The outlook and potential applications for these AMMs are discussed critically, highlighting the barriers to overcome to enable widespread use of AMMs in industry, with recommendations for promising manufacturing methods to progress the field of microscale acoustic metamaterials.AMMs are artificial structures designed to achieve novel material properties that can manipulate acoustic waves in ways beyond the scope of natural materials. For example, AMMs have been documented with the following unusual features: negative bulk modulus; negative effective density; negative refractive index; and sub-wavelength diffraction/focusing ,20. WhilLong before the term “metamaterials” was coined, there existed optical devices that demonstrated these extraordinary material properties. Arguably, the earliest conceived “metamaterial” was created in 1898 and was composed of jute fibres twisted in opposing directions . This stJust prior to Pendry’s artificial microstructure research, Helmholtz resonator arrays were among the first AMMs with potential applications discussed. In 1992, Sugimoto proposed using Helmholtz resonators embedded in a train tunnel to suppress shockwaves . PhononiA significant breakthrough in AMMs is the development of acoustic cloaks. Working similarly to an optical cloak, an acoustic cloak can shield an object from the surrounding acoustic field. A feasible 2D acoustic cloak prototype was first published in 2008 , buildinAMMs have been separated into three distinct categories for the purposes of this review. These categories are (i) periodic/lattice metamaterials; (ii) coiled structures and metasurfaces; and (iii) active metamaterials. These classifications are not exclusive and there may be some overlap, with some AMM examples belonging to multiple categories.This type of AMM consists of periodic structures of repeating cells or meta-atoms. Phononic crystals describe a variety of lattice structures with fluid, elastic or combination inclusions in a different material matrix . HoweverPhononic crystals, despite their dimensional limitations, are still effective at sound attenuation applications. This is presented in a known phononic crystal design , which hPeriodic, locally-resonant AMMs can be produced in a variety of shapes and sizes, and from different materials, manufactured using both 3D printing and conventional fabrication techniques. The local resonances can be created using two different kinds of meta-atom: intrinsic and inertial ,62. IntrInertial meta-atoms operate as independent mechanical oscillators and can be mathematically modelled using a dynamic system of masses, springs and dampers. This allows accurate resonant behaviour prediction of increasingly complex, periodic metamaterials. Some inertial examples built upon existing intrinsic designs by adding coatings to spherical inclusions , while oMembrane-based acoustic metamaterials are capable of producing near complete reflection in a narrowband at low frequencies . In theiThis sub-section is concerned with AMMs with chiral and coiled structures. These structures often, but not always, coincide with metasurfaces—defined as planar metamaterial and hence operating in 2D only. Given that these coiled structures are asymmetric, using two dimensions considerably simplifies the design. The coiled nature of these materials is inherently suitable for subwavelength devices, as propagating waves are made to travel much further than the global length of the structure.An example of this type includes labyrinthian channels of air, and there is evidence that such a design proved capable of directing a phase shift in acoustic wavefronts . Figure Another quality achievable by this AMM type is negative refraction . This caCoiled structures have also exhibited significant benefits in enhancing the broadband abilities of AMMs . A gradiThis category includes controlled, time-variant AMMs, meaning their material properties, physical structure or acoustic effects can be altered during use and are not constant variables. These materials can be modulated using a variety of external inputs and can function either with or without exchanging energy gain from the external acoustic field . This brA common feedback method in active AMMs involves using piezoelectric material to convert acoustic pressure into electric current and vice versa. Initial active AMM designs consisted of rudimentary fluid cells separated by piezoelectric boundaries . Figure Other actuation methods for active AMMs include mechanical energy, temperature and applied magnetic fields. Helmholtz resonator-based devices can incorporate mechanical plungers to change their internal cavity volume, resulting in a shift in acoustic properties. To illustrate, The spectral effects of temperature have been analysed and applied to create a tuneable array of water-filled Helmholtz resonators . By adjuHydrogels are prevalent materials in the literature surrounding active AMMs, broadening the field by introducing soft, flexible capabilities that allow for easier physical actuation. These malleable, hydrogel-based AMMs are referred to as metagels. A novel hydrogel-composite design, shown in By using hydrogels as a primary design material, this also allows for movement control through changes in hydration. A novel hydrogel-composite metamaterial uses a horseshoe-shaped unit design to facilitate the unusual behaviour of shrinking with water absorption, and vice versa . The micA relevant lattice-type structure here is tensegrity metamaterials, which, through their geometrically non-linear response, can tune global elastic characteristics and hence modulate the structures effect on acoustic waves. These structures consist of an assembly of rigid, compressive elements and deformable, tensile elements  that resActive AMMs are designed to have modifiable effects on the surrounding acoustic field; therefore, the changeable parameters are usually directly related to the equations for acoustic wave propagation. Bulk modulus and effective density are of specific interest, with a significant portion of active AMM research focused on allowing these parameters to be tuneable ,99,100. The previous section demonstrates the wide range of AMM designs in the literature but is by no means exhaustive. However, it is clear that the concepts and theoretical approaches are being pushed towards greater complexity, and it is our opinion that the greatest barrier to increased complexity will be the ease of manufacture. In this section, we will discuss additive manufacturing methods currently used, which, within the scope of this review, include vat polymerization techniques, powder bed fusion techniques, extrusion and deposition methods, and hybrid techniques.The distinctive feature of vat polymerization is that targeted photopolymerization is employed to construct custom 3D parts, as opposed to other methods which use heat-based fusion or material extrusion. These additive manufacturing techniques can be broadly defined as the curing of photo-reactive polymers via an electromagnetic radiation source  . The tecThis 3D printing technique was among the first developed and involves the scanning of a photo-curable liquid, layer by layer, to create a 3D part . TraditiIn another example of SLA, a small-scale Helmholtz resonator  was successfully fabricated and validated experimentally with a commercially available SLA device , the minFurther SLA restrictions exist in the printable material options, which generally are a form of photocurable liquid; however, there is still versatility, with ceramic inclusions being used to improve mechanical, thermal, electrical and magnetic properties of the base resin . AdditioSLA printers have been utilized to fabricate devices for acoustic and ultrasonic applications. Notably, an in-air ultrasonic beam shifter was produced from clear resin, using a commercially available 3D SLA printer (Asiga PICO2 HD), where the prototype was experimentally verified to confirm its beam shift performance on acoustic fields . The acoMulti-material micro-SLA has also recently been demonstrated, with several coloured resins used in a single print and a layer thickness of 30 μm , illustrFrom SLA technology, another microscale printing method emerged—digital light processing. This is similar to SLA; however, DLP cures an entire layer at once by projecting patterns of light . This meNotably, a micro-beam was fabricated using DLP with a layer thickness of 30 μm and an exposure time of 3 s (per layer) . The priIn the literature, SLA and DLP are further contrasted, with SLA’s printing speed  restricted to several mm/h due to its stepwise regime, where each individual layer takes seconds to manufacture and is 50–100 μm thick. Unlike SLA, DLP’s printing is generally continuous and restricted instead by resin cure rates and viscosity, and hence can achieve speeds at least two orders of magnitude faster . A gyroiThe printable material is limited by similar restrictions to SLA; additionally, multi-material prints are more difficult to achieve with DLP because of the holistic nature of the curing mechanism. One of the earliest DLP multi-material prints, in 2011, required the material vat to be removed and replaced to print a different material . This meA subsequent multi-material DLP method, with a top-down approach, employed an air jet-based cleaning step to minimize resin waste and cross-contamination . This noThere are currently promising advancements in printing metallic materials with DLP by using silver nanoparticles incorporated in a polymer matrix . AnotherAn emerging tomographic technique building on DLP technology employs a single-step multi-beam exposure, omitting the typical stepwise protocol of most additive manufacturing methods . This unFirst proposed in 1997 , multi-p3 cube of replica shale rock with a minimum feature size of 160 nm [MPP in prior studies has repeatedly demonstrated nanoscale print size, succeeding the aforementioned vat polymerization methods significantly in ultra-high resolution ,125. An f 160 nm . The priIn another example of MPP fabrication, The manufacture of small-scale lattice structures is a significant area of interest for tensegrity metamaterial research. Discussed earlier in While the printable resin needs to be photocurable, nanocomposite inclusions can be added that allow for a variety of material properties, such as conductivity, magnetism, piezoelectricity, mechanical properties, high refractive index  and biodVat polymerization methods are prolific for constructing AMM prototypes, with several examples presented in the previous AMM section in Ability to mass-produce:In terms of vat polymerisation techniques, DLP is the fastest production method by a significant margin. Additionally, its simultaneous layer curing regime also allows for the creation of several printed parts without additional printing time. SLA is the next fastest, due to its layerwise scanning, with MPP being the slowest fabrication process because of its precise point-by-point curing mechanism. Unlike DLP, printing extra parts adds proportionally to the printing time for SLA and MPP. Considering this, DLP is the most promising vat polymerisation method for mass-producing AMMs.Minimum printable feature size:MPP has the highest resolution of all the additive manufacturing techniques discussed in this review, with a minimum feature size well within the nanoscale (as low as 60 nm). It also is not restricted by the use of support structures, as the point-by-point polymerization regime can fabricate at any 3D spatial point, unlike DLP and SLP, which are limited to a 2D build surface. DLP performs better than SLA at printing small features, but with the resolution of both generally restricted to the microscale. DLP has been recorded to reach minimum features sizes as small as 600 nm. With all systems, the build volumes are typically inversely correlated with build volume, meaning higher resolution comes at the cost of smaller finished products.Multi-material printing:Multi-material printing is inherently difficult with vat-polymerisation techniques due to the possibility of cross-contamination of the different liquid resins used as build material. Despite this, several multi-material prints were presented in this section, with SLA presenting the best multi-material capabilities in Active metamaterial fabrication:The construction of active metamaterials is closely linked to the range of build materials available to the applicable fabrication methods. Printed active metamaterial examples using vat polymerisation methods are sparse in the literature, with no AMM-specific examples mentioned in this text. The material restrictions of the photocurable liquid resin hinder the fabrication of actuatable metamaterials, making it generally unfeasible to produce these with any vat polymerization techniques. The exception to this is hydrogel-based active designs, as these can be quite easily fabricated using SLA, DLP and MMP, with an SLA-printed example shown in Powder bed fusion is an additive manufacturing method wherein a heat source is applied to a bed of fine particles in order to bind that construction material together and form highly-detailed 3D parts . The sco2 laser heats the layer, point by point, until the desired pattern is sintered together. The platform descends in the Z direction, and a new layer of powder is spread over evenly and the process repeats until the part is finished. SLM operates a similar way but with some important differences. In order to melt the material, the laser must be higher energy, also requiring inert gas to function. Overall, SLM has several benefits over SLS, namely it is faster and has a lower minimum layer thickness (20 μm for SLM compared to 80 μm for SLS). SLS can print with nylon, thermoplastic and polystyrene, while SLM can print with metallic powders. Recent developments that have incorporated optical fibres with high-powered lasers have enabled further material options for SLM, with composite and ceramic printing now within reach. Both SLS and SLM can yield high quality prints; however, post-processing is required to eliminate potential issues such as porosity, surface roughness and crack defects. SLM can incur welding defects, so it requires thermal post processes to assure part quality.These two powder bed fusion methods use laser beams to bond powder into 3D parts. However, there is a key distinction between SLS and SLM. The former works solely with plastics, whereas the latter primarily with metals . The manSLS can print with relatively high spatial resolution and short print duration; however, SLS generally requires one of these qualities to be prioritized over the other. An example of this is perforated plates for tuneable AMMs , as showAn example of an SLS-fabricated AMM in the literature is a locally-resonant panel structure that demonstrates acoustic band gaps . This stSLS can also be used to print soft AMMs with mechanical actuation capabilities. Several configurations of a 3D soft auxetic metamaterial were fabricated using SLS, as shown in SLM is one of the most promising 3D printing methods for fabricating metallic small-scale parts without the use of composite materials, resins or binders . Key parRecent studies have been conducted on the design and testing of lattices fabricated by SLM ,147. In A novel application of printed AMMs is ultrasound cloaking technology, designed to mask the presence of objects from the surrounding acoustic field. SLM has been used to construct an AMM underwater cloak with an operational bandwidth of 100–900 kHz, frequencies commonly used in medical ultrasound and non-destructive testing (NDT) . This brThis innovative technique builds on the traditional SLS method and shows promising improvements in some key printing variables . While tMJF was used to print an acoustic metamaterial panel capable of attenuating sound at a specific frequency range . The MJFThe internal resonance of the system is very sensitive to small deviations in geometry, which is a common problem in the fabrication of resonant AMMs. Therefore, refining the accuracy of powder bed fusion printing methods could be a significant step towards the large-scale production of feasible locally-resonant metamaterials for industrial application.This fusion method uses an electron beam as the heat source in order to fabricate both metal and plastic material formulas . The powA recent example shows EBM used to fabricate a metallic micro-lattice with mechanical capabilities that can replicate bone . Both EBGiven the powder bed can only contain one fusible material at a time, multi-material parts are usually comprised of one base powder  and another non-fusible support powder/material. A novel multi-material design was proposed consisting of an auxetic chiral meta-cell that is permeated with silicon to make a hybrid material . The chiPowder bed fusion methods are less prevalent in AMM research than that of vat polymerization; however, there is potential in the discussed methods  to contribute to the improved fabrication of AMMs. Namely, MJF shows significant capability in the fast production of polymer-based AMMs with improved mechanical properties and resolution on par with SLS. With further development, MJP could not only surpass other powder bed methods in mechanical part quality and minimum feature size but shows potential to exceed many vat polymerization and extrusion techniques as well. Below, the variables pertaining to the additive manufacture of AMMs for use in widespread industry are discussed, with the aforementioned techniques compared and contrasted for suitability in each category.Ability to mass-produce:MJF is the fastest production method presented in this sub-section, as it employs infrared projections to render an entire layer at once. This feature is not shared by the other powder bed fusion methods listed and is very useful for the batch production of AMM devices. The other methods employ a scanning regime for heating parts, meaning decreasing the layer thickness to achieve finer features will have a detrimental impact on the printing time. SLS and SLM examples are shown with printing speeds in the X–Y plane of 4000 mm/s and 1200 mm/s, respectively. An added benefit for AMM manufacture is these techniques require minimal support structures.Minimum printable feature size:The minimum feature sizes of all powder bed methods are roughly equivalent to each other, ranging from >400 μm (EBM) to 100–20 μm (SLM). The minimum feature size is heavily restricted by the particle size of the build powder, with the particle size always being smaller than the resulting layer thickness. Refining powder to nanoscale particle sizes would be expensive and not necessarily successful in increasing method resolution . Overall, powder bed methods are limited to microscale features—SLM presents the smallest feature size of 20 μm.Multi-material printing:In general, powder bed methods cannot yet create multi-material parts without additional processing/material addition after printing, with an example if this shown in Active metamaterial fabrication:SLS and MJF utilize plastics as the build material, which is generally more appropriate for fabricating standard AMMs, whereas SLM uses metal particles. Only SLM and EBM have metal-printing capabilities, which could be used to fabricate active metamaterials controlled by electromagnetic fields. Hydrogels are not compatible with any powder bed techniques, limiting the actuation possibilities of printed metamaterials.Extrusion-based techniques were among the first additive manufacturing techniques developed, with a significant literature surrounding this form of 3D printing in macro-scale. This technology is capable of multi-material and multicolour prints with a wide range of materials, such as plastics, ceramics , metals This method extrudes build material through a nozzle via a system using pneumatic or mechanical actuation . The keyA novel freeform extrusion technique derived from the principles of FDM has been used to fabricate high-density ceramic lattices . The parAnother emerging FDM method involves the liquid deposition of a conductive nanocomposite, comprised of multiwall carbon nanotubes suspended in PLA, using a high volatility solvent as the dispersion medium . This meThis method uses similar principles to FDM; however, the key difference is this method employs inks as the construction material . Unlike DIW is known to have a wide variety of ceramic and polymer material options, whereas current metallic material options are largely restricted to ink with silver particles . Small-sTypically, DIW techniques have a resolution strongly limited by their nozzle diameter; however, submicron feature sizes have been achieved using support structures and microcapillary nozzles of varying diameters . A holloDIW technology can use a variety of ink formulations with different materials, including live cells and human tissue mimics. A novel printer set-up bio-printed thick (>1cm) vascularized tissues within 3D perfusion chips, as displayed in DIW has been established as a high-resolution technique for a broad array of material options and with minimal cost . Its liqIntroduced in the previous DIW section, inkjet printing ejects ink onto a substrate using either controlled thermal or piezoelectric/mechanical actuation . In the An application of interest for inkjet manufacturing methods is the production of acoustic metamaterial sound absorbers. An additive polyjet system was used to fabricate a thin-walled (1 mm thick), periodic AMM, with resonance exploited in the unit-cells by coupled tubes . The geo6 m/s2. The layerwise regime of this method can stack up to 2000 sub-micrometre layers in one second, resulting in a printing speed three to four orders of magnitude faster than comparable methods with equivalent minimum feature sizes. An added benefit of electrohydrodynamic jetting is that nozzle clogging at the submicron scale is not an issue, unlike FDM, enabling nanometre-sized jets (less than 100 nm) to be used. The speed limits of this jetting technique are currently 0.5 m/s in-plane and 0.4 mm/s out of plane, and the resolution is currently limited to 100 nm (dependent on fibre thickness and jet apertures). This technology can be expanded to print with different ink compositions, including other polymers, biomaterials and living cells.An innovative manufacturing method uses precisely-controlled electrohydrodynamic (EHD) jets to achieve submicron feature sizes with extremely fast printing speeds . Figure Extrusion/deposition methods are an extremely robust and versatile branch of additive manufacturing. This is reflected in its application in AMM manufacture  and acroAbility to mass produce:FDM has the cheapest build material (solid filament) and likely the fastest production method due to the continuous material deposition regime. With DIW, small volumes of ink droplets are added discontinuously, leading to a slower print rate; however, jetting systems partially addresses this limitation. This is evident in the print rate of thermally-actuated jetting systems, which can be as high as 160,000 droplets per second. Some electrohydrodynamic setting systems can reach quick print rates of 0.5 m/s in-plane and 0.4 mm/s out of plane.Minimum printable feature size:The ink droplet-based methods offer extremely small printable feature capabilities, with resolutions of 2 μm to 600 nm for DIW and 10 μm to 100 nm for jetting systems. The minimum feature size has been greatly reduced by recent innovations in both these methods, such as integrated laser annealing for metallic DIW printing , and empMulti-material printing:In general, extrusion/deposition techniques have strong multi-material printing capabilities because instead of reacting with a large volume of build material, in a vat or bed, the part is built up using controlled addition of material. FDM has particularly strong multi-material printing capabilities because its solid filament has minimal risk of cross contamination and can easily be interchanged during printing. Ink-droplet methods also demonstrate these abilities, with a complex multi-material example presented in Active metamaterial fabrication:Although no active examples were provided in this section, the potential of these methods to achieve this is substantial. The printed examples consisting of ceramic  or metalA novel hybrid technique combined digital light processing (DLP) and direct ink writing (DIW) to produce multi-material prints with functional inks, including liquid crystal elastomers (LCEs) and conductive silver inks . This unAnother DLP-based process allows microscale features to be applied to macro-scale 3D objects, pre-printed with any suitable manufacturing method . This prWhile this example only demonstrates hydrophobic effects, a variety of surface properties could be induced with an appropriately designed microstructure. This DLP-based process can be used alongside virtually every additive manufacturing method and has the potential to revolutionize how surface effects are added to 3D-printed parts. Manipulating microdroplets in this fashion shows potential for use in several important applications, such as oil spill clean-up, micro reactors, 3D cell cultures and self-cleaning devices.2 and thulium lasers are used in conjunction with a DLP chip to achieve the simultaneous melting of both pre-heated polymers in the desired layer shape. Adding DLP principles to the SLS/SLM manufacturing process has enabled simultaneous laser beam melting (SLBM) to be developed . This teA polyjet approach incorporating inkjet technology alongside the photocuring of resin, a distinctive feature of vat polymerization methods, has been applied to manufacture novel Luneburg lenses for the purposes of ultrasound beam steering . The smaThe field of acoustic metamaterials holds much promise for the future, with novel solutions to acoustic problems becoming increasingly close to real-world applications. While the field of AMMs covers such problems as earthquake engineering, building acoustics and underwater applications, we have chosen to focus on the potential uses of acoustic metamaterials on airborne systems for audio and ultrasonic applications.With airborne sound covering wavelengths from metres to millimetres, the scale of materials that interact with these sounds is similar, so not only are novel subwavelength meta-atom designs important, but also the ability to create them is crucial. Additive manufacturing has proved to be the most promising out of existing technologies, exceeding its subtractive counterparts, to build up complex materials from their constituent meta-atoms. However, the outlook is challenging for translating this academic research into fully-realized devices for industrial and consumer applications. Rapid prototyping, while cheap for everyday use, still becomes expensive with scale, particularly techniques requiring custom materials. Additive technologies working at multiple scales is not feasible, so obtaining micron resolution over scales in excess of a centimetre is close to impossible. Complex shapes are challenging, and aspects of temperature dependence or curing/annealing mechanisms of raw materials can mean that it is difficult to print with adequate accuracy and reliability. For example, the printing of membranes—commonly used to provide mechanical behaviour to a meta-atom—is challenging, where at a small scale the raw materials can have post-production tension and warping that is difficult to compensate for. In addition, each technique has production requirements unique to itself. As mentioned, vat polymerization techniques cannot print voids or inclusions directly, instead requiring vents or other ways to leak out the original liquid polymer. Designs of AMMs need to account for that, providing another barrier to production.Clearly, for a practical and realisable AMMs, we need to be able to create large-scale materials (and ultimately devices) with small-scale features. Additive manufacturing is the best candidate for both rapid prototyping and bulk manufacture, but limitations on specific fabrication characteristics act as a barrier to AMM research, constraining printable ideas and restricting its potential. Given that metamaterials typically derive their properties from repeating units of local resonators or geometrical features, a key issue in their manufacture is consistency and reproducibility across length scales. A material in which some of the meta-atoms failed to print correctly could alter the acoustic properties and potentially disrupt the predicted acoustic behaviour. This high dependency on uniformity, or reliability of shape, across meta-atoms means that AMMs are still very challenging to create, even with additive manufacturing, and especially at smaller scales. The robustness of metamaterials to meta-atom failure could be a key area of research to progress AMMs to commonplace use.This review discussed many viable additive manufacturing methods for small structures, and it discussed techniques best suited to address some key fabrication parameters for AMMs. MPP was found to have the smallest printable feature size, followed by DIW, jetting systems and DLP, with the highest resolution of these methods well within the nanoscale. This result is supported by similar findings in the literature ,169. ForOther noteworthy techniques include SLM and EBM, which excel at fabricating metallic parts with microscale resolution without the use of support structures. Vat polymerisation and ink deposition methods are the set of techniques best suited to printing hydrogels—a key material for the fabrication of some active metamaterial designs. Vat polymerisation shows strong potential for AMM fabrication, with SLA, DLP and MPP all being robust techniques with individual benefits; however, their main limitation lies in their material restrictions and post-processing requirements. Further development to overcome these challenges would be beneficial for their application in AMM construction.Over the following 10 years, the next generation of practical AMMs will need to come from either significant progress in rapid prototyping and manufacturing technology, or the discovery of meta-atom designs that have complex physical behaviour while being sufficiently simple enough to be printed. Active acoustic metamaterials promise to push development forward, compensating for manufacturing challenges by adding complexity through controlled actuation rather than static structure, but they are also difficult to produce beyond very basic systems at the smallest scale. Nevertheless, it will be exciting to monitor which of these two strands of research will succeed first.


Apolipoprotein D is a chordate gene early originated in the Lipocalin protein family. Among other features, regulation of its expression in a wide variety of disease conditions in humans, as apparently unrelated as neurodegeneration or breast cancer, have called for attention on this gene. Also, its presence in different tissues, from blood to brain, and different subcellular locations, from HDL lipoparticles to the interior of lysosomes or the surface of extracellular vesicles, poses an interesting challenge in deciphering its physiological function: Is ApoD a moonlighting protein, serving different roles in different cellular compartments, tissues, or organisms? Or does it have a unique biochemical mechanism of action that accounts for such apparently diverse roles in different physiological situations? To answer these questions, we have performed a systematic review of all primary publications where ApoD properties have been investigated in chordates. We conclude that ApoD ligand binding in the Lipocalin pocket, combined with an antioxidant activity performed at the rim of the pocket are properties sufficient to explain ApoD association with different lipid-based structures, where its physiological function is better described as lipid-management than by long-range lipid-transport. Controlling the redox state of these lipid structures in particular subcellular locations or extracellular structures, ApoD is able to modulate an enormous array of apparently diverse processes in the organism, both in health and disease. The new picture emerging from these data should help to put the physiological role of ApoD in new contexts and to inspire well-focused future research. ApoD, identified and named almost 50 years ago, is a protein belonging to the Lipocalin family. Experimental research on ApoD has been accumulating, encouraged by numerous findings of ApoD relationship to many human diseases, from cancer to cardiovascular, metabolic or neurodegenerative conditions. This affluence of scientific reports has described many aspects of ApoD functional features, but a fundamental question remains to be responded: does ApoD moonlight, performing different biochemical functions in different biological contexts? or does it display a distinctive biochemical role that is being used in several physiological systems?Along this half-a-century of ApoD research many reviews focused on this protein have been published. All of them are narrative in nature and many concentrate on specific details of ApoD such as its relationship to disease, often underlining partial and not critically assessed views on many aspects of ApoD biology.Aiming at answering the central questions posed above, we have performed a systematic review of all primary research published until January 2021 where ApoD properties have been investigated in the chordate phylum. Reports were tagged and classified according to their contributions to “molecular properties,” “gene data,” “regulation of expression,” “disease-related,” “cellular trafficking,” “tissue and organ function,” and “protein physiology.” The level and quality of experimental evidence were critically evaluated to try to identify cause-effect relationships. The picture emerging from this approach should help to understand the physiological role of ApoD and to inspire well-focused future research.n = 216), Regulation of Expression , Gene Data , Molecular Properties , Cellular Trafficking , Tissue and Organ Function , and Protein Physiology . To assess the current knowledge on the physiology of ApoD, we performed a literature review of primary publications in a systematic manner, searching the National Library of Medicine database with the PubMed engine . Using the search query “Apolipoprotein D” OR Apo-D OR ApoD, we recovered 851 entries from which 39 narrative reviews were excluded. Following a Title/Keywords screen, we selected 787 entries for further assessment. Following exclusion criteria we finally selected 417 articles reporting research on the Lipocalin ApoD in chordates as the final review sources, and stored them in a Zotero (v5.0.88) collection. The complete collection is available in the Following full article reading, we classified each tagged report with subheading terms to guide the organization of the review. We then performed an evaluation of the conclusions statements of each report based on technical and argumentative consistency, according to existing state-of-the-art standards and required experimental controls. In cases of uncertainty, experts in each field were contacted and asked for their objective judgement.in silico prediction platforms and tools were used in this work: ProtParam (https://web.expasy.org/protparam/); DeepLoc-1.0 (http://www.cbs.dtu.dk/services/DeepLoc/); Gene Ontology database (http://geneontology.org/); Human Protein Atlas (https://www.proteinatlas.org); Mouse gene expression (http://www.informatics.jax.org/expression.shtml); miRNA database (mirdb.org). The ApoD multiple sequence alignment was generated with ClustalX2 , and the 3D structure of ApoD was visualized with ViewerLite 4.2 (https://chemweb.ir/accelrys-viewerlite/). A model of HApoD with sugars attached was built with GlyProt (http://glycosciences.de/modeling/glyprot/php/main.php).The following databases and ApoD is an early-diverging member of the Lipocalin family, with its phylogenetic origins traced back to the origin of chordates , a potential for oligomerization, and an association with lipids. The predicted acidic isoelectric point  implicatReference Collection 1, References contributing to this section are listed in The ApoD 3D crystal structure has been solved for the human protein after modification of several residues that rendered the protein prone to aggregation. The unique human Cys116 is close to one of the hydrophobic loops, and was also mutated to facilitate crystallization. The structure reveals a typical Lipocalin fold Skerra,  composed2O2. Dynamic information extrapolated from the crystal structure has allowed further modeling of ApoD binding to small ligands, lipoprotein particles or membranes. These studies help to understand a methionine-dependent lipid antioxidant mechanism (see below) and to study the influence of glycosylation on these functional properties. In addition, the ApoD monomer crystal structure, combined with modeled glycosylation conformations, was used to generate coherent models for the conformations of ApoD oligomers  or small-angle X-ray scattering (SAXS) in solution, interesting conformational changes elicited by ligand binding were detected, resulting in further ordering of the already stable Lipocalin fold. ApoD structure is also stable upon protein oxidation with Hligomers  later coReference Collection 2, References contributing to this section are listed in in silico studies of human ApoD revealed no interference of sugars with binding pocket access. As mentioned above, sugars were soon revealed to be linked to ApoD, with a relevant carbohydrate contribution (~15–22%) to its apparent molecular weight. Two asparagine sites were experimentally demonstrated to be glycosylated, and Interesting variations of ApoD carbohydrate moiety have been reported between species , between various tissues in a single species , within a tissue, or between health and disease conditions. Patterns of glycosylation have also been demonstrated to be sex-dependent . For example, less glycosylated forms of ApoD are present in mouse/human brain tissue compared to plasma, with differences in both terminal sialic acid and core N-linked oligosaccharides. A clear increase in α2-3 sialoglycosylation of plasma ApoD distinguishes, with high sensitivity, children with Autism Spectrum Disorder from healthy controls. Within a single tissue  there is also variegation in the degree of ApoD sialylation. These variations generate size and charge heterogeneities with potential functional consequences worth exploring.Reference Collection 3, References contributing to this section are listed in in vivo , as demonstrated with recombinant ApoD, mutated at specific Met residues, and produced in a human cell line. Oxidation-dependent ApoD dimerization is resistant to guanidine hydrochloride (GuHCl) but not to urea, indicating that it is based on non-covalent intermolecular bonds. This property has allowed to detect ApoD dimers in specific brain regions of Alzheimer's disease patients  that also correlate with disease progression. In contrast, GuHCl extracts from healthy control brains show only monomeric forms of ApoD.in vitro protein oxidation with H2O2. The putative contribution of intermolecular disulfide bonds in the tetramer (involving human Cys116) has not been explored.A tetrameric stable form of native ApoD in BCF, but not in plasma or CSF, has been demonstrated and characterized by multi-angle laser light scattering, analytical ultracentrifugation, HDX-MS and SAXS. Experimental data using progesterone as a ligand and the native ~100 kDa ApoD tetramer from BCF, supports a particular tetramer conformation among those predicted by molecular modeling, where the binding pocket opening is accessible and the sugar moieties do not interfere in the inter-subunit interface . MonomerReference Collection 4, References contributing to this section are listed in Reference Collections 5, 6, The ability to bind progesterone was a defining feature of the most abundant protein in BCF, therefore named progesterone-binding cyst protein (PBCP) or gross cystic disease fluid protein 24 (GCDFP-24). Later on, this protein was demonstrated to be identical to ApoD purified from plasma HDL particles. Ligand-protein interaction at the ApoD binding pocket induces conformational changes leading to a more ordered structure, but does not result in major structural changes or altered oligomerization. These dynamic changes, though subtle, might have implications for ApoD interactions with other proteins or lipoprotein particles. Ligand binding reports are grouped in in vitro ligand-binding experiments have been performed with the human protein, using either recombinant ApoD (expressed by bacteria or eukaryotic cells) or native protein purified from BCF or plasma HDL. Arachidonic acid (AA) shows the highest affinity, while various AA derivatives  show no binding by Trp-fluorescence titration.Progesterone accommodation in the pocket involves a tryptophan residue heavily conserved in the Lipocalin family , a male axillary precursor of odorants, was identified by gas chromatography-mass spectrometry (GC/MS) after temperature/pH switch and chloroform extraction from purified ApoD.Interestingly, various ligands  whose interaction with ApoD has been demonstrated by a different technique, do not alter Trp-fluorescence, raising the possibility of other sites of interaction. Molecular dynamics simulations infer flexible binding of oxidized derivatives of AA  around the conserved Met93 at one of the hydrophobic patches at the entrance of the pocket. This particular form of lipoperoxide binding to ApoD is not expected to produce changes in fluorescence of Trp-127, located at the bottom of the binding pocket. A proof of interaction is experimentally supported by site-directed mutagenesis combined with HPLC-detection of reduced lipids (HETEs) after exposure to ApoD. This interaction underlies the antioxidant activity of ApoD (see below). Interactions of ApoD to higher-order lipid structures, like lipoprotein particles or cellular membranes, are particularly relevant since they determine the range of sites and biological contexts where ApoD function can be performed. They might depend on protein-protein or protein-lipid contacts.As mentioned above, co-purification of ApoD with LCAT might indicate the potential for a protein-protein interaction in nascent HDL particles, but a clear demonstration of ApoD-LCAT complex is not available. In contrast, a clear protein-protein interaction does account for human ApoD presence in HDL particles. An intermolecular disulfide link between ApoD Cys116 and ApoA-II Cys6 has been demonstrated by peptide digestion followed by sequencing and mass spectrometry (MS). This interaction, however, is an exclusive property of human ApoD due to its unique unpaired cysteine.A putative disulfide-linked ApoD-ApoB100 complex was also proposed, but evidence is based on predictions from electrophoretic mobility in reducing/non-reducing conditions and immunoblot detection with anti-ApoD antibodies only, or with antibodies raised against LDL particles. An almost full characterization of 23 out of 25 cysteine residues in ApoB-100 by MS-analysis and peptide sequencing found no bonds with ApoD , the intracellular domain of the Leptin Receptor (OB-Rb), the transmembrane glycoprotein Basigin (BSG), and the Scavenger receptor class B type 1 (SRB1).Other potential interactions of ApoD have been explored with classic two-hybrid systems, where protein-protein contact takes place in the cell nuclei or cytoplasm, both requiring ectopic expression of ApoD in non-native biological compartments unsuitable for disulfide linked proteins (see sections Protein Structure and Cellular Trafficking). Alternatively, co-immunoprecipitation in vivo conditions. Other candidate ApoD receptors (LDLR and CXCR-4) are predicted from physiological contexts, where downstream consequences of ApoD exposure are modified by antagonists of these receptors. However, a direct interaction with these receptors has not been explored.The weak interaction reported between ApoD and the intracellular domain of OB-Rb, combined with its presumed topology within the cell, should discard this finding as a biologically relevant interaction for ApoD unless it is replicated. For membrane proteins such as BSG and SRB1, proposed as putative membrane receptors for ApoD, co-localization by confocal imaging is often used as additional evidence. However, protein-complexes are below the resolution of standard co-labeling techniques, and methods relying on distance-dependent energy transfer, super-resolution or immunoelectron microscopy would be desirable as further evidence in relevant Reference Collection 7, References contributing to this section are listed in 3 particles . Analysis of HDLs separated by electrophoretic mobility in non-denaturing PAGE followed by in-gel trypsinization, identified ApoD within the HDL-α2 type, in a 1:100 ratio with respect to ApoA-I. The presence of ApoD in HDLs has been confirmed also in human CSF and in baboon and mouse plasma. Additionally, plasma ApoB-100 positive LDL particles contain ApoD as well, but at lower concentrations (~8 ng ApoD/μg LDL vs. ~69 ng ApoD/μg HDL3). The generalized interaction with different lipoparticles in several species suggests that ApoD-lipoparticle interactions must rely on a mechanism independent of ApoD-ApoA-II disulfide bond, a human HDL rarity. The fact that ApoD-LDL interaction is prevented by detergents, and do not take place with recombinant ApoD where hydrophobic surface residues have been mutated , suggests a hydrophobicity-dependent ApoD-lipoparticle binding mechanism  further demonstrates its ability to bind to lipidic structures without requiring a protein-protein interaction. These unilamellar vesicles represent a simplified version of the outer phospholipid layer of HDLs, LDLs or a membrane bilayer . In addiThe knowledge accrued on ApoD protein structure, its glycosylation and oligomerization properties, as well as its interactions with small ligands and other lipidic structures are relevant for its physiological roles in lipid management, and should help to get a global picture of how these molecular properties are put to work in various physiological contexts.Reference Collection 8, References contributing to this section are listed in The gene coding for ApoD locates in an autosomic chromosome that shows ample synteny in chordates  and metabolic stress (serum deprivation). Also, the transactivator TAp73 mediates ApoD expression upon cell differentiation. The mouse ApoD promoter region has been recently assessed experimentally, and an alternative promoter region has been related to OS-induced ApoD expression.DNA methylation, inferred from the CpG content of the gene promoter region, is also an important regulatory mechanism for ApoD transcription, with an inverse relationship between level of DNA methylation and ApoD gene transcription. This gene regulation mechanism has been shown in different physiological or pathological contexts: in esophageal, colorectal and astrocytic cancers, in the expression profile defining Th17 lymphocytes, and for the androgen receptor-response in male sexual development.Reference Collection 10, References contributing to this section are listed in The mRNA 3′-UTR is known to influence its stability and translation efficiency. ApoD 3′-UTRs show a high degree of conservation in mammals, and display shorter lengths and higher G+C content than those observed in average mammalian gene UTRs. These differences have been proposed to underlie a tight regulatory control of ApoD translation. In this context, a number of miRNAs have been predicted to control ApoD translation, possibly by binding to the 3′-UTR. Some of these miRNAs, like miR-229b-3p, miR-423-3p, and miR-490-3p, have been experimentally tested and implicated in the post-transcriptional downregulation of ApoD expression in rat male reproductive system upon metabolic dysfunction.in silico analyses of these 5′-UTR variants in mouse and human ApoD show upstream initiation codons, upstream open reading frames, and predicted secondary structures that suggest a tight control on ApoD gene expression.The 5′-UTR of ApoD also presents relevant properties for the regulation of ApoD expression. It is rich in short-tandem repeats (STR), specifically in primates. Long stretches of STRs are predicted to affect transcription and translation, which might have contributed to the neurodevelopmental changes that underlie primate evolution. Furthermore, mammalian ApoD genes show several alternative 5′-UTRs forms, possibly arising from alternative splicing. The alternative 5′-UTRs of the mouse ApoD gene have been experimentally tested and shown to underlie differential protein expression in several mouse tissues, with a particular 5′-UTR variant being strongly induced upon OS. Moreover, Reference Collection 11, References contributing to this section are listed in In terms of genetic variation for the ApoD gene, over 4,600 variants have been found in the GRCH38.p12 (annotation Release 109) assembly of the human genome, while 187 are reported in the short variants (dbSNP) and structural variants (dbVar) databases. Six variants that involve missense, intron insertions and 3′-UTR insertions, are predicted to involve molecular consequences. Some of these variants have been linked with variable support to human cancer, metabolic or neurological diseases  tag for this systematic review  . The celAvian egg fluids are also rich in ApoD, with the interesting property that egg white ApoD positively correlates with egg freshness.Reference Collection 13, References contributing to this section are listed in Reference Collections 14–16; The analysis of tissue expression pattern leads to a general conclusion: in spite of its wide distribution, ApoD is never ubiquitously expressed, never in all cell types in a tissue, or at all times in a given cell type. ApoD is expressed in most tissues with a salt-and-pepper spatiotemporal pattern, suggesting a fine control that depends on particular physiological cell states. Furthermore, all tissues bear ApoD-expressing cells and cells able to endocytose ApoD protein from the extracellular milieu (see section Cellular Trafficking). These expression features, along with ApoD being a very stable protein, result in a high protein abundance when measured in high-throughput analyses, and in a lack of exact fit between mRNA and protein expression in a given tissue or cell . Upregulation of ApoD by androgens is well documented in different preparations like breast explants, male genital fibroblasts or primary epithelial cells from male axillary apocrine glands. This regulation is mediated by nuclear androgen receptor (AR), and ApoD is being used as an AR activity assay . These patterns of response can be extended to the many disease situations reviewed in section ApoD-Disease Relationships. In addition to the abundant correlative data from human diseases, experiments in animal models of disease analyzed in vivo, primary cell cultures and cell lines support a major conclusion: ApoD is a key player in the endogenous response to a variety of potentially harmful stimuli. The damage and stress responsive p73/p63 and JNK pathways have been demonstrated to up-regulate ApoD  remains to be elucidated. Not all stressful conditions trigger ApoD expression . Retinoic acid induction of ApoD expression, mediated specifically by RARα in breast cancer cells, correlates with the anti-proliferative action of this signaling pathway. However, the potential role of ApoD in regulating cell division (see section Protein Physiology) must be dependent on the physiological/pathological context. For example, in the model of pericyte-endothelium interactions mentioned above, mural cells decrease ApoD expression upon interaction with endothelial cells, when they would stop dividing to generate mature capillary structures.Finally, confluency and senescence in cell cultures also trigger ApoD expression . A clear negative correlation between ApoD expression and malignancy has been found in nervous system tumors, fibrosarcomas, breast, colorectal, hepatic, renal and cervical cancers. The general association of a good prognosis with high ApoD expression strongly suggests a protective anti-tumoral function for this Lipocalin. While studies of prostate cancer have not evidenced unambiguously such a pattern, some studies show regional ApoD expression differences  that are still compatible with a defensive tissue response to neoplastic transformation. ApoD tumor-suppressing activity has been experimentally tested and an inverse relationship between ApoD promoter methylation, ApoD expression and outcome is supported by various reports . WhetherCardiovascular and metabolic diseases (particularly diabetes) as well as infection or injury, are also accompanied by ApoD upregulation. Again, OS might be a common link to ApoD response to these diseases, for instance in atherosclerotic plaques depending on disease progression, or upon oxidative degradation of glycated proteins in diabetes.In contrast to the many diseases where ApoD expression changes have been reported, few genetic variations of ApoD have been widely or robustly linked to disease risk or prognosis , the signal peptide removal in the protein sorting process, the N-linked oligosaccharide modification carried out in RER-Golgi, and a secretion of the mature glycoprotein to the extracellular environment in several tissues and cultured cells. All these data make ApoD a typical soluble extracellular protein undergoing a canonical secretory pathway, a consensus attained by subcellular localization prediction algorithms and data present in gene ontology databases (see Methods). Additionally, subcellular traffic of ApoD can also lead to its exportation out of the cell in different formats that include ApoD tetramers, HDL-associated ApoD and extracellular vesicle-associated ApoD .in vitro interaction assays that were designed for proteins naturally occurring in the cytoplasmic or nuclear compartments (like the classic two-hybrid assays) preclude the finding of functionally relevant interactions for ApoD. An alleged cytosolic ApoD would likely be non-glycosylated and improperly folded in the absence of its intramolecular disulfide bonds.However, several reports have interpreted their findings about ApoD biological roles on the basis of protein partitioning in cytoplasm and/or nuclear compartments. Aside of technical issues questioning those results, some reports use overexpression of fusion-tagged proteins, which are known to undergo unnatural compartmentalization or degradation. Also, strategies based on A number of studies have shown the presence of ApoD in RER and vesicular compartments of different eukaryotic cells. Immunoelectron microscopy (EM) of nervous system cell types has unambiguously identified ApoD in the outer nuclear membrane-RER membrane complex and in lysosomes. Lysosomes isolated from placental cells, monitored with biochemical techniques, contain ApoD. Likewise, clathrin-coated vesicles isolated from hen's ovaries also contain ApoD.Recently, the subcellular location of ApoD has been studied in detail in basal conditions and under experimental stimuli, either by immunogold-EM microscopy, or by fluorescence immunocytochemistry combined with established cell compartment markers and monitored by well-documented standardized confocal microscopy. These studies have detected the presence of ApoD in RER, the Golgi apparatus, endosomes, lysosomes, autophagosomes, multivesicular bodies, as well as in the outer side of plasma membrane, clathrin-coated vesicles and caveolae. The protein does not localize to mitochondria or peroxisomes, and has not been immunolocalized inside cell nuclei. Lysosomal ApoD localization has been demonstrated in astrocytes, oligodendrocytes, Schwann cells, fibroblasts, and neurons.Although those experiments were performed at a fixed time point, serial-time experiments demonstrated that the presence of ApoD in the endosome-lysosome compartment is stable and dynamically enriched upon OS exposure. Long-lasting lysosomal location of ApoD depends on its glycosylation and hydrophobicity, as mutated bacterial recombinant ApoD is maintained in lysosomes only transiently. Targeting of ApoD to the lysosomal compartment occurs not only in ApoD-expressing cells, but also in non-expressing neurons upon exposure to exogenous ApoD or when co-cultured with astrocytes. The fact that ApoD is a stable component of subsets of lysosomes at a given time, connects many of the apparently diverse physiological roles of ApoD (see section Protein Physiology).Reference Collection 33, References contributing to this section are listed in It is undoubtedly established, as discussed above, that ApoD is being secreted to the extracellular environment following a canonical secretory pathway . HoweverA third mechanism by which ApoD becomes extracellular is by traveling in HDL particles. ApoD-HDL association can take place during HDL biogenesis, or ApoD can associate to HDLs while the lipoparticles bind to cell membranes and perform their lipid efflux activity. However, these mechanistic details and the particular subcellular origin of the HDL-associated ApoD detected in body fluids need to be investigated. The plasma membrane location of both ApoA-I dependent HDL biogenesis , as well as the internalization of ApoD by cells cultured in the presence of its native or recombinant forms, are the experimental basis supporting the endocytosis of this Lipocalin. It takes place both under control conditions and in response to specific biological stimuli. ApoD endocytosis appears as a general property of this protein, as it has been reported in birds and mammals. Particularly, in glia-neuron co-cultures ApoD is found to be exclusively transported in EVs from astrocytes to neurons, where it gets internalized. The current view of several extracellular formats of ApoD  makes it worth to study whether different membrane interaction mechanisms or endocytosis paths are used for ApoD internalization.https://www.proteinatlas.org/ENSG00000189058-APOD/cell). Whether ApoD-membrane association is mediated by protein-protein or protein-lipid interactions requires further research (see sections Protein-Protein Interactions and Binding to Lipid-Rich Structures). ApoD association to the extracellular side of the plasma membrane is coherent with both, its traffic from RER to plasma membrane by the canonical exocytotic path and with its cell contact before internalization. ApoD-plasma membrane interaction has been experimentally demonstrated and is currently considered an established localization for ApoD in human cells . Both in embryonic development and during the remodeling process of wound healing, ApoD increased expression is causally linked to undifferentiated mural cells migration, though is not consistently related to cell proliferation . A crosstalk between endothelial cells (ECs) and MCs governs the switch of the angiogenic cellular process from a proliferative/migratory state to a differentiation state, characterized by quiescence and adhesion. This switch is essential for vessel morphogenesis. Blood vessel angiogenesis involves reactive oxygen species (ROS), EC-derived PDGF-BB, JAGGED1 and NO, as well as MC-expressed NOTCH3. These secreted and cell contact-mediated signaling downregulates ApoD in MCs, a process linked to blood vessel maturation. Experimental manipulation of ApoD levels demonstrates that ApoD regulates the adhesion of MCs to the extracellular matrix, and low levels of ApoD promote Zyxin- and Vinculin-positive focal adhesion contacts. Concordant effects have been reported in retinal choroid vessels: ApoD-KO mice show neovascularization with increased permeability.Myocardial cells show slight expression of ApoD under normal circumstances, but the tissue surrounding an injured/infarcted area promptly upregulates ApoD. The study of this process in ApoD-KO mice indicates that ApoD is associated to protection from cell death in the injured tissue. This ApoD protective role on cardiomyocytes is dependent on a proper protein fold and strongly correlates with its antioxidant activity (see sections Binding to Lipid-Rich Structures and Protein Physiology). In this experimental paradigm, the protective activity is attained by increasing ApoD in plasma, though it is unclear whether ApoD levels are also elevated in the infarcted tissue. We thus propose that ApoD function in myocardial tissue protection and remodeling might be based on: (1) A modulation of cell viability in cardiomyocytes and vessel ECs, possibly due to internalization of plasma-derived ApoD, and/or (2) a regulation of cell differentiation related to the angiogenic response described above, organized by ECs and MCs.Reference Collection 36, References contributing to this section are listed in in vivo by using two different ApoD-KO mouse lines and a transgenic mouse (hApoD-Tg) driving the expression of human ApoD under the control of the human THY1 gene. This hApoD-Tg mouse ectopically expresses hApoD mostly in neurons, but the protein is present in plasma and other organs physiologically relevant to metabolism. Also, adenovirus-driven liver production of mouse ApoD has been used as a paradigm of acute overexpression, leading to elevated protein levels in plasma.The role of ApoD in metabolism has been analyzed in vivo. However, more work is needed to derive definitive evidence for understanding the role of ApoD in lipid and carbohydrate metabolism. So far, the relevant results indicate that the functional relationship of ApoD with various metabolic parameters is, at most, indirect and dependent on other physiological conditions.The metabolic consequences of altering ApoD levels have been evaluated mostly in plasma and liver, although also in retina, and measured in a variety of experimental settings: fasting or non-fasting conditions, different feeding diets, and different sex or age of animals. No clear pattern can be extracted for the role of ApoD on carbohydrate metabolism, where reports describe varied outcomes on glucose tolerance or insulin resistance depending on experimental conditions. Some consistency is observed in the effects on triglycerides (TG): Loss of ApoD leads to decreased hepatic TG content and increased plasma TG, while overexpression leads to elevated TG levels in liver and unaltered or decreased triglyceridemia (depending on the strategy used for ApoD overexpression). On the other hand, variations in plasma cholesterol levels are also reported, with various outcomes upon ApoD loss or overexpression. Also, association of ApoD polymorphisms have been found with both increased and decreased HDL-cholesterol species. The finding of ApoD being able to mediate binding of HDL to LDL, and of HDL particles to actively dividing carcinoma cells, suggests that it can regulate lipid traffic indirectly by influencing lipoparticle dynamics. Variations in local physiological contexts of this traffic mechanism might contribute in very different ways to the final systemic outputs measured in the experimental settings studied Reference Collection 37, References contributing to this section are listed in Bone cells, from bone marrow stem cells (MSCs) to osteoblasts, are reported to express ApoD in cell culture systems , 17, andReference Collection 38, References contributing to this section are listed in As presented above, the current evidence supports a general view in which non-neuronal cells become the source of ApoD in response to different stimuli, and neurons count on the Lipocalin for its cellular functions by internalizing ApoD. Neurotransmission is one of those functions modulated by ApoD. Analysis of downstream effects in gene expression in the brain of ApoD-KO or hApoD-Tg mice, reveal an enrichment of genes related to synaptic transmission. Particularly, changes in glutamate, somatostatin, dopamine and acetylcholine neurotransmission have been independently documented by receptor binding assays, HPLC determination of neurotransmitters or their catabolites, or receptor immunodetection. These effects might underlie the behavioral phenotypes related to locomotor function, motor and spatial learning, and retinal function observed in mice with altered expression levels of ApoD.ApoD addition to cultured neurons results in neuritogenesis and synaptogenesis, which are crucial during neuronal development and underlie neuronal plasticity of established circuits. Neuritogenesis is promoted in immature neurons in culture by the combined addition of ApoD and retinoic acid in the absence of serum. Experiments combining ApoD addition with receptor antagonists indicate that ApoD-dependent neurite extension can be mediated by LDLR, and possibly also by CXCR4 activation, pathways known to be involved in neuronal differentiation.Another general function of ApoD, extensively analyzed in loss-of-function and transgenic mice, is its role in the glial response to dyshomeostatic changes in the nervous system due to oxidative, metabolic or traumatic stresses. Many studies have reported an acute regulation of ApoD expression under these insults, either experimental or triggered by disease (see sections Regulation of Expression and ApoD-Disease Relationships), supporting an overall neuroprotective role now widely accepted as a functional label for this Lipocalin. Both astrocytes and oligodendrocytes express and secrete ApoD in response to stress. The protein exerts an autocrine and paracrine neural tissue protection, which results in functional preservation of OS-challenged dopaminergic systems, of neurons affected by kainate excitotoxicity or suffering from Aβ-related degeneration. Astrocytes, although not an abundant source of ApoD in basal conditions, quickly respond to OS with a JNK-dependent expression of ApoD, which is secreted to the extracellular milieu as cargo on the surface of extracellular vesicles . The proA long-lasting homeostasis maintenance role for ApoD has been also proposed in the process of physiological aging of the nervous system, where this protein has been shown as the most consistently overexpressed in primates and rodents. Also, life-expanding strategies in model organisms, like caloric restriction, promote ApoD expression not only in the nervous system but also in cardiac and skeletal muscle . The homin vitro-demonstrated ApoD ligands, The predominant expression of ApoD in myelinating cells under control conditions  has prompted experimental studies, using cultured primary cells and ApoD-KO and hApoD-Tg mice, that clearly support the implication of ApoD in the myelination process during development, in the lifelong maintenance of the myelin sheath, and in the remyelination that occurs in response to environmental insults. These processes have been analyzed in the mouse brain corpus callosum as well as in the peripheral sciatic nerve. ApoD is required for a proper and timely response to a crush injury in PNS nerves, helping to recover locomotor function. ApoD promotes myelin clearance and regulates angiogenesis and macrophages recruitment to the wound site, processes that are essential for subsequent axonal regeneration and remyelination. ApoD contributes to optimize myelin clearance, carried out by transdifferentiated Schwann cells and infiltrating macrophages, through two complementary actions: control of lipid-mediated inflammatory signaling and optimization of the phagocytosis process itself. Data indicates that ApoD regulates and control the tissue levels of AA and lysophosphatidylcholine  and of the regulatory Fyn kinase. This mechanism requires preservation of lysosomal membrane integrity (see section Protein Physiology).Reference Collection 39, References contributing to this section are listed in In this final section we aim at discussing the available knowledge, derived from state-of-the-art research critically assessed in this review, to give a plausible answer to the central question posed in the Introduction: In order to achieve its pleiotropic roles, does ApoD moonlight between different biochemical functions when expressed in different contexts, or instead ApoD displays a distinctive biochemical role that works on varied physiological situations?The presence of ApoD in extracellular formats such as lipoprotein particles and exosomes evidences its ability to associate to higher-order lipid structures. According to our systematic search no unambiguous evidence exists for the secretion of protein monomers in native conditions. Non-denaturing electrophoresis analysis of CSF revealed only high-molecular weight ApoD oligomers, while in plasma it has been repeatedly identified in lipoprotein particles preparations. In the particular case of BCF, where an extremely high concentration of ApoD is produced, the protein assembles in tetramers through protein-protein interactions. Finally, ApoD secreted by cultured astrocytes is internalized by neurons only if the conditioned extracellular media has not been depleted of extracellular vesicles. A protein region encompassing the first three β-strands, highly conserved in chordates , and sevThese results shed doubts on a view of ApoD widely cited  as a “lipid transporter,” a task that a secreted globular monomer could easily achieve.Unquestionably though, it is the ability of ApoD to bind small hydrophobic ligands of varied shapes inside its β-barrel pocket. However, when free ligands  have been mechanistically related to ApoD function, binding data are compatible with a buffering or quenching function, or a very local shuttling of the ligand at the most, rather than to a generalized long-range ligand transport between cells. A curious case is the expression of ApoD in feather follicles of pheasants, only in skin areas with specific plumage colors, suggestive of a pigment-retention function. Similarly, the ligand bound to ApoD in sweat from human axilla could be the source of slowly released volatile odor molecules. These ligand-retention functions compare well with that of crustacean ApoD homologs, also linked to their carapace coloration , a LCAT reaction product that exerts a negative feedback on LCAT activity. By quenching LPC, ApoD would maintain LCAT activity over a wide range of LPC product concentration. This specific LPC quenching function is compatible with the small amounts of ApoD recovered from HDLs, since only a transitory presence of ApoD might be needed when LCAT is adding cholesterol to the lipoparticle. As for the HDL-LDL interaction (see section Roles of ApoD in Metabolism Regulation), the putative consequences of ApoD presence in HDLs on cholesterol management in the organism would therefore be of an indirect nature, and could explain the lack of correlation between ApoD and cholesterol content in many physiological or pathological situations.in vitro biochemical assays, cellular experimental systems, and in vivo experiments with animal models where ApoD expression was manipulated. Moreover, further validation for this role comes from experimental approaches testing the expression of human ApoD in evolutionary distant organisms. Overexpression of human ApoD in Drosophila increases lifespan in both normal and pro-oxidative experimental conditions. Also, replacement in plants of the native chloroplast Lipocalin (LCNP) by human ApoD, targeted to thylakoids, rescues drought and OS sensitivity of the mutant. Lipid peroxidation control is evidenced in both reports as the mechanism mediating the organism response.A fundamental advance in defining ApoD molecular function was its role in organismal protection against OS, achieved by a control of the magnitude of lipid peroxidation, measured at tissue or cellular levels. This role has received strong experimental support from An ApoD antioxidant mechanism has been demonstrated using oxidized AA-derivatives in solution or auto-oxidized liposomes. ApoD is able to reduce free radical-generating lipid hydroperoxides to inert lipid hydroxides. In this reaction, the residue Met93 exposed on one of the surface hydrophobic patches of the protein  is conveThe direct antioxidant activity of ApoD and its demonstrated stable location in the lysosomal compartment put forward a new view of ApoD protein physiology that holds high explanatory power in the understanding of a number of apparently varied ApoD roles.ApoD control of redox state can be performed directly on both, cell membranes and lipoprotein particles. The unilamellar vesicles where ApoD-reducing activity has been demonstrated are a good experimental model for both types of lipid-based structures. Lipid peroxide products are mainly derived from cellular membranes, which are a major target for cell-generated ROS. The ability of ApoD to keep low levels of membrane-originated lipid peroxides, together with the positive correlation of ApoD content in HDLs with their antioxidant capacity, and the promotion of HDL-LDL interaction by exogenously-added native ApoD, support the protective action of ApoD in both types of lipidic structures (membranes and lipoparticles). Additional evidence comes from the existence of ApoD insect homologs stably anchored to cell membranes  supports this notion. Plasma membrane modulation is also coherent with the observed correlation of ApoD content in HDLs and their ABCA1-dependent cholesterol efflux capability in macrophages, or the subtle changes in lipid content in lipoprotein particles of subjects with ApoD polymorphisms. Through its influence on membranes and lipoparticle dynamics, without a need of binding cholesterol, ApoD can modulate its flux within and between cells.Additionally, the lysosome is a “cell death/survival controller” by its fundamental recycling, detoxifying and proteostatic functions. Lysosomal ApoD would condition whether a failure in the lysosomal compartment takes place upon a wide array of disease/injury situations, thus contributing to the final cell fate. This ApoD-dependent cell fate decision can be extended to developmental processes as well.Finally, a role of ApoD in innate immunity has been frequently reported, while no mechanistic link to the protein physiology was proposed. We suggest that ApoD, with its lysosomal optimization mechanism, can modulate the efficiency of phagocytic cells, like it has been demonstrated in injury-recruited macrophages, therefore influencing many of the maintenance and immune responses of the organism.This view makes us to propose that ApoD lipid-binding properties are more related to management of lipid-based structures composition (membranes or lipoparticles) and a control of their redox state, than to lipid transport. Whether similar membrane-stabilizing properties endow ApoD-positive exosomes with resistance properties to be efficient cargo transporters in disease or tissue damage situations, would be worth studying.A different aspect of ApoD physiology scarcely studied is the role of its demonstrated N-linked glycosylation, which has been proven to be tissue and species specific, and to be essential for both, ApoD interaction with lipoparticles and for its cellular localization in the endolysosomal compartment. This is particularly important because of the association between redox signaling and glycan profiles, which in turn could affect several signaling pathways . Findings in Drosophila reveal that loss of an ApoD homolog alters PI3K association to the plasma membrane (Hull-Thompson et al., vs. monomeric forms of ApoD underlie its managing function on cell membranes or lipoparticles.To test whether oligomeric To characterize the extent of ApoD redox cycle, maintaining antioxidant ApoD activity thanks to the intervention of reductases, and the implications of a potential upper limit to this mechanism due to ApoD oligomerization. This aspect can be key to fully understand ApoD function in aging and disease.To analyze the effects of differential glycosylation on ApoD interactions and functions.Recent studies on a Drosophila homolog (Yin et al., At a more general tissue/organ level, several functions are relevant to be studied in more depth, such as the ApoD role on feather and skin physiology, neuronal synaptic function, and metabolism.Finally, it is worth to analyze the potential exchange between the nervous system and systemic pools of extracellular ApoD in its different formats, not only to fully understand its roles in the organism, but also for a potential therapeutic use of ApoD in nervous system diseases.Reference Collection 1 (McConathy and Alaupovic, Reference Collection 2 (Eichinger et al., Reference Collection 3 (McConathy and Alaupovic, Reference Collection 4 (Blanco-Vaca and Pownall, Reference Collection 5 (Haagensen et al., Reference Collection 6 (Fielding and Fielding, Reference Collection 7 (Utermann et al., Reference Collection 8 (McConathy and Alaupovic, Reference Collection 9 (Drayna et al., Reference Collection 10 (Lambert et al., Reference Collection 11 (Provost et al., Reference Collection 12 (Drayna et al., Reference Collection 13 (Holmquist, Reference Collection 14 (Drayna et al., Reference Collection 15 (Bouma et al., Reference Collection 16 (Bouma et al., Reference Collection 17 (Simard et al., Reference Collection 18 (Loerch et al., Reference Collection 19 (Do Carmo et al., Reference Collection 20 (Blais et al., Reference Collection 21 (Suresh et al., Reference Collection 22 (Provost et al., Reference Collection 23 (Tew et al., Reference Collection 24 (López-Boado et al., Reference Collection 25 (Provost et al., Reference Collection 26 (Simard et al., Reference Collection 27 (Boyles et al., Reference Collection 28 (Balbín et al., Reference Collection 29 (Wiklund et al., Reference Collection 30 (Curry et al., Reference Collection 31 (Bujalska et al., Reference Collection 32 (Albers et al., Reference Collection 33 (Bouma et al., Reference Collection 34 (Simard et al., Reference Collection 35 (Vieira et al., Reference Collection 36 (Sarjeant et al., Reference Collection 37 (Desai et al., Reference Collection 38 (Martineau et al., Reference Collection 39 (Hildebrand et al., Reference Collection 40 (Steyrer and Kostner, The original contributions presented in the study are included in the article/MG and DS: conceptualization, writing, review, and editing. Both authors contributed to the article and approved the submitted version.This work was supported by a Ministerio de Ciencia e Innovacion grant PID2019-110911RB-I00 to MG and DS.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The goal of immuno-oncology is to potentiate a durable antitumor immune response. The immunosuppressive tumor microenvironment (TME) presents a substantial challenge to current systemic therapies due in part to a lack of available tumor antigen, dense stroma, an abundance of immunosuppressive cells and cytokines, and poor antigen presentation. A multimodal approach that combines intratumoral immunotherapy with tumor ablation addresses several of these challenges. In this review, we evaluate the current data regarding this promising therapeutic approach.Several intratumoral immunotherapeutic agents have shown efficacy in controlling local disease; however, their ability to induce a durable systemic immune response is limited. Likewise, tumor ablation is well-established due to its role in local disease control but generally produces only a modest immunogenic effect. It has recently been recognized, however, that there is potential synergy between these two modalities and their distinct mechanisms of immune modulation. The aim of this review is to evaluate the existing data regarding multimodality therapy with intratumoral immunotherapy and tumor ablation. We discuss the rationale for this therapeutic approach, highlight novel combinations, and address the challenges to their clinical utility. There is substantial evidence that combination therapy with intratumoral immunotherapy and tumor ablation can potentiate durable systemic immune responses and should be further evaluated in the clinical setting. Immuno-oncology (IO) is a cornerstone of modern cancer therapy. Most notably, checkpoint inhibitors (CI) are now first-line treatment for a variety of metastatic solid tumors, and their role in the adjuvant and neoadjuvant settings is expanding. There are, however, some limitations to existing systemic IO, as efficacy is largely dependent on an antigen-specific effector T-cell-mediated response that may not occur in the setting of a poorly antigenic tumor. Effector cells must also overcome several physical and biochemical obstacles in order to exert their effector function. Dense tumor stroma, an inhibitory chemokine/cytokine profile, and the presence of a variety of immunosuppressive cell types contribute to a harshly immunosuppressive tumor microenvironment (TME). These barriers are not easily surmounted with checkpoint inhibitor therapy alone. There is now widespread interest in developing new therapeutic modalities that can transform a cold, immunosuppressive TME into one that is immunogenic .Human intratumoral immunotherapy (HIT-IT) is one promising strategy that contends with the drawbacks of systemic IO while addressing the TME directly ,3. HIT-ITumor ablation is another modality that has gained recent interest for its potential to induce an antitumor immune response. Ablative techniques vary in mechanism; however, their therapeutic efficacy is primarily achieved via local tumor destruction. Ablated tumor tissue is subsequently left in situ where inflammatory mediators, damage-associated molecular patterns (DAMPs), and other immunomodulatory factors are released . ImportaAs the immunomodulatory effects of tumor ablation have been better defined and novel HIT-IT are developed, it has become clear that these modalities could have unique synergistic potential  8]. In . In 8]. Oncolytic viruses have been in various stages of investigation for over a century. While these agents were initially investigated for their direct oncolytic potential, recent interest has centered on their ability to induce antitumor immunity. T-VEC, a genetically modified herpes simplex type 1 virus, was the first oncolytic virus to gain FDA approval (2015) and is indicated in the treatment of advanced melanoma. T-VEC has been shown to induce regression of injected tumors and can potentiate an immune-mediated response in non-injected lesions; however, it is ineffective for control of distant disease . VariousSun et al.  investigMultimodal treatment with oncolytic viruses and tumor ablation is theoretically counterproductive, as ablative therapies are designed to destroy tumor cells while oncolytic viruses require host tumor cells to be living in order to replicate. Investigators have attempted to address this challenge in novel ways. Yamada et al.  used a nAdoptive cell immunotherapy (ACT) has shown clinical efficacy in treating hematologic malignancies and is under active investigation for use in solid tumors. Multiple late-phase clinical trials are underway to evaluate tumor-infiltrating lymphocyte therapy (TILS) in metastatic melanoma, and other cell-based immunotherapies are in early trials. Of the many challenges limiting the usefulness of ACT in treating other solid tumor types, several of these may be addressed by combining intratumoral injection of immune cells with tumor ablation. First, cells delivered intravenously must traffic to and infiltrate tumor tissue to exert their effector functions, which is a process that is often inhibited by poorly immunogenic tumors. Immune cells delivered intratumorally can circumvent the physical barrier created by dense tumor stroma, do not need to traffic hematogenously, and could be delivered in sufficient quantities to overcome immunosuppressive signaling in the TME . Tumor aApproaches that combine intratumoral ACT with tumor ablation have been studied in the preclinical setting. Machlenkin et al.  used cryp = 0.003), though overall survival was not significantly different. Notably, DCs were harvested from patients’ peripheral blood, incubation and expansion took only one week, and the treatment was well tolerated by patients. Another study showed similar antitumor immune responses with direct IT injection of OK432 in a breast cancer RFA model [Other groups have expanded on the intratumoral DC and ablation strategy by investigating agents that can augment antigen processing and effector T cell activation. Udagawa et al.  briefly FA model . It remaWhile there is a clear theoretical basis for combining ablation with APC-based therapies, the role of other cell-based therapies is not well defined. Few studies have evaluated non-APC immune cells in combination with ablation. In a phase I study, Lin et al.  investigPattern recognition receptors, primarily expressed by antigen-presenting cells, have long been known for their role in regulating the inflammatory response to pathogens. PRRs act to recognize both conserved pathogen-associated molecular patterns (PAMPs) in the setting of infection as well as endogenous cellular damage-associated molecular patterns (DAMPs) released by stressed or dying cells. Upon recognition of PAMPs or DAMPs, PRRs potentiate a wide variety of immunologic effects including cytokine release, APC differentiation, antigen presentation, and lymphocyte activation. Several vaccine adjuvants have been successfully developed that take advantage of these immunostimulatory properties . There hThere is clear synergistic potential in combining intratumoral PRR agonists with ablation. By delivering PRR agonists to the site of ablation, they may function as an in situ vaccine adjuvant, potentiating the immune response to antigen released by ablation. Behm et al.  evaluateAlthough TLR agonists are promising agents to combine with tumor ablation, they can cause notable off target effects. Various TLRs are known to be expressed on certain tumor types and T cell subsets. Signaling through receptors on these cell types can promote tumor proliferation and immune evasion. Additionally, variable downstream effects are seen dependent on the type of TLR targeted. For example, TLR4 agonism enhances proliferation and activity of immunosuppressive regulatory T cells, while TLR1/2 and TLR8 agonism reduces the suppressive effects of Tregs . BalanciOther PRR pathways are being evaluated for their therapeutic potential. STimulator of INterferon Genes (STING) is an intracellular PRR that recognizes cytosolic DNA and stimulates the production of type 1 interferons. The STING pathway plays a central role in endogenous antitumor innate immunity primarily through host immune cell ingestion and recognition of tumor-derived DNA. Activation of this pathway induces production of type 1 interferons which regulate widespread local and systemic immune functions including DC maturation, T cell priming, chemotaxis, and further cytokine production . STING aSynthetic biomaterials are increasingly being studied for use as immune modulators and many of these novel materials are uniquely suited to local delivery. Several of these agents have been studied as adjuncts to tumor ablation. Zhou et al.  developeSmaller molecule synthetic biomaterials have also been evaluated in combination with ablation. Chen et al.  combinedIntratumoral cytokine injection has also been studied in combination with tumor ablation. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a cytokine that, among other functions, activates APCs and has been shown to trigger a systemic antitumor response. This cytokine is a critical part of the mechanism of action of T-VEC, which incorporates a GM-CSF cassette, causing local expression in injected tumors . InteresMultimodality therapy with HIT-IT and tumor ablation presents unique opportunities and challenges. Substantial progress has been made in investigating the efficacy of this therapeutic approach in the preclinical setting, yet few translational studies have been completed thus far and results of those that have been completed have been underwhelming. Questions remain regarding the role that this approach will play in increasingly complex and effective IO treatment paradigms that are emerging. Additionally, many promising current IO therapies have been woefully understudied for their use as HIT-IT or in combination with ablation. As new IO agents are developed, it will be important to consider their potential for use as HIT-IT or in combination with ablation, especially if systemic delivery is found to be toxic or infeasible.There is obvious potential for therapies to be developed de novo to exploit the individual mechanism of action of each ablation technique and the unique biologic properties of the resultant ablated tumor. For example, thermal ablation tends to induce necrotic cell death, which produces significantly more inflammation, immunostimulatory cytokine release, and available tumor antigen compared to the apoptosis induced by IRE ,48. SomeWhile avoidance of systemic toxicity is a key advantage of local therapy with HIT-IT and ablation, the addition of systemic adjuncts such as checkpoint inhibitors could further enhance the resultant systemic immune response induced by this therapeutic strategy. Checkpoint blockade has proven to be of limited efficacy in poorly immunogenic tumors, especially tumors of an immune desert phenotype . These tThere are several challenges to implementing combined HIT-IT and ablation therapeutic approaches . MelanomAnother practical challenge to studying combination HIT-IT and ablation is that most patients undergoing visceral tumor ablation are doing so for curative intent. Patients that have no evidence of viable disease after treatment would lack the in situ untreated metastatic lesions that are often used to evaluate anenestic response to HIT-IT. It would be exceedingly challenging to determining radiographic response in a primary tumor that has been both injected with HIT-IT and ablated, especially given the changes that are typically seen radiographically after tumors are ablated. itRECIST criteria  were devA multimodal approach with HIT-IT and tumor ablation addresses many of the major limitations of current IO-based therapies, and has the potential to induce a durable antitumor immune response. Further studies, especially of a translational nature, are needed to determine which patients, which combination of modalities, and in which tumor types this approach is most beneficial.