Myocardial infarction (MI) is one of the leading causes of heart-related deaths worldwide. Following MI, the hypoxic microenvironment triggers apoptosis, disrupts the extracellular matrix and forms a non-functional scar that leads towards adverse left ventricular (LV) remodelling. If left untreated this eventually leads to heart failure. Besides extensive advancement in medical therapy, complete functional recovery is never accomplished, as the heart possesses limited regenerative ability. In recent decades, the focus has shifted towards tissue engineering and regenerative strategies that provide an attractive option to improve cardiac regeneration, limit adverse LV remodelling and restore function in an infarcted heart. Acellular scaffolds possess attractive features that have made them a promising therapeutic candidate. Their application in infarcted areas has been shown to improve LV remodelling and enhance functional recovery in post-MI hearts. This review will summarise the updates on acellular scaffolds developed and tested in pre-clinical and clinical scenarios in the past five years with a focus on their ability to overcome damage caused by MI. It will also describe how acellular scaffolds alone or in combination with biomolecules have been employed for MI treatment. A better understanding of acellular scaffolds potentialities may guide the development of customised and optimised therapeutic strategies for MI treatment. Myocardial infarction (MI) is responsible for major cardiovascular associated death worldwide . FollowiIn this regard, formerly tissue engineering approaches have paid attention to leverage stem, precursor or differentiated CM cells-based approaches to resolve and restore infarcted myocardium and ventricular remodelling post-MI ,8. It haScaffolds are therapeutic materials whose specific composition can bring forth biological cues along with mechanical support; they include bioscaffolds, patches, matrices and hydrogels. The basic framework of scaffolds is synthesised from polymers whose physical and chemical properties can be easily controlled. However, such scaffolds alone possess low bioactivity and therefore their major functional benefits are limited to structural and mechanical support only.Cell-based approaches coupled with scaffolds have improved to some extent the efficacy of transplanted cells in terms of retention but some concerns still exist: limited nutrient and oxygen supply to the cells loaded on the scaffold due for instance to an insufficient vascularisation as well as the risk of host tissue immune response that can compromise cell viability ,10. MoreOn the other hand, functional acellular scaffolds coupled with biological molecules, drugs or other physiologically useful components are endowed with the potentiality to orient towards normal physiological function. They may encompass the key to unlock and elicit endogenous repair and regeneration processes at the infarcted site. Depending on their structural and functional targeted role, these acellular scaffolds can be architected to impart useful characteristics which include biodegradability, biocompatibility, biomimetism in addition to represent carriers and recruiters of useful endogenous and exogenous therapeutic molecules . To reduThis review summarises the studies conducted in the past five years on acellular scaffolds as therapeutic candidates to overcome post-MI ventricular remodelling. Indeed, a comprehensive understanding of this promising therapeutic approach can pave its path to clinical translation.Acellular scaffolds with biological, polymeric or bio-artificial nature, endowed or not with electrical cues, have been developed and tested both in vitro and in vivo .ECM provides structural support to cardiac tissue and is also involved in the integration of extracellular signals and the regulation of morphology, migration, differentiation and proliferation of cardiac cells ,16. AfteAcellular scaffolds with physiological anisotropy and integrated bioactive ECM can be potential candidates to provide a favourable and conducive microenvironment to recruit host cells and restore normal function . The strThe therapeutic potential of decellularised porcine myocardium slices (dPMS) as acellular scaffolds was investigated in a rat MI model by Shah & Zhang group. dPMS scaffolds with different thicknesses (300 and 600 μm) were applied to the infarcted myocardium. In both groups, dPMS scaffolds were tightly affixed to the myocardium that provided strong LV mechanical support and impeded thinning of the LV wall. Histological analysis demonstrated that dPMS was endured as a whole patch unified with host myocardium with no gap. Moreover, it also contributed effectively to host cell infiltration, formation of vessels and improvement in cardiac function. At 4 weeks, in comparison to 300 μm thickness dPMS, 600 μm patch was more effective in preventing thinning of LV wall and fractional shortening (FS). In addition to this, the 300 µm thickness patch degraded significantly as observed after 4 weeks of transplantation thus highlighting that the durability of dPMS can be compromised in long-term applications . Unlike Porcine small intestine submucosa ECM (SIS-ECM) is a decellularised ECM structure derived from small intestinal submucosa that encompasses several useful and clinically operative characteristics. SIS-ECM has retained its native 3D structure and cell signalling cues that provide a favourable and encouraging homeostatic atmosphere to allow functional survival and retention of host cells. Moreover, it is absorbable and has preserved sufficient biological activity to regulate the endogenous host cells infiltration, differentiation, growth and proliferation. It shows another appealing feature that consists of the inability to provoke host immunological responses ,29. CorMThe relevance of using an intact bioactive scaffold for post-MI functional recovery was highlighted by the comparison between CorMatrix-ECM and its biologically inactivated form. The latter was obtained by treating the bioactive scaffold with either glutaraldehyde cross-linking or by guanidine hydrochloride. Both bioactive and inactivated CorMatrix-ECM scaffolds were implanted in a rat MI model. The results confirmed that bioactivity within ECM acellular scaffold is crucial for its functional impact. In fact, after 6 weeks, it was shown that only bioactive ECM could promote vasculogenesis and repair. Moreover, in this study through in vitro assays, it was demonstrated that human cardiac fibroblasts and fibroblast growth factor-2 (FGF-2) efficiently interact with ECM scaffolds thus suggesting that the in vivo functional impact of ECM on vasculogenesis and stimulation of pro-repair processes, including enhanced activation of epicardial progenitor cells, could be mainly attributed to these endogenous components. Despite these interesting results, however, the authors pointed out that the chemical procedure of CorMatrix-ECM decellularisation caused cell toxicity . The cliVentriGel is an ECM injectable hydrogel that is derived from decellularised porcine myocardium. Unlike other biological scaffolds that acted as mechanical support, the VentriGel was designed for in situ assembling into a fibrous and porous scaffold that provides an ECM microenvironment, to recruit endogenous cells. In terms of stiffness, it is weak as it has two-fold lower stiffness than healthy myocardium, yet this feature allows its less invasive delivery through a catheter ,35. A saSeveral cardiac ECM therapies exploit adult animal cardiac tissues to derive ECM. Adult mammalian cardiac tissues, however, possess limited regenerative potential. In contrast, the cardiac tissues of neonates can undergo favourable and robust cardiac regeneration. It is therefore hypothesised that ECM derived from neonatal tissues might be more effective therapeutically in comparison to adult tissue-derived ECM. A study in an adult mice acute MI model compared the therapeutic potential of ECM derived from neonatal mouse heart (nmECM) versus ECM derived from adult mouse heart and concluded that nmECM can more effectively improve cardiac function and averts widespread adverse LV remodelling .In recent years, placenta and amniotic membrane (AM) are used in surgical regenerative medicine, originally because they are a good source of juvenile cells . It has During the fabrication of scaffolds, specific architectural, biochemical and biophysical characteristics can be acquired through customisation of various factors such as mechanical properties, microstructure and cross-linking degree. For bioengineering applications, degradable polymers are employed frequently . In recePoly-(ε-caprolactone) (PCL) is a synthetic FDA approved semi-crystalline aliphatic polyester with high thermal stability, biocompatibility, long-term biodegradability and elastomeric properties. Poly-(glycerol sebacate) (PGS) is a synthetic thermoset polymer well-known for its elastic, biodegradable and biocompatible characteristics that make it a useful candidate for soft tissues engineering and cardiac scaffold construction . When elCyclic SAPs to promote activated hydrogel scaffold gelation in in vivo rat MI model. This simple approach has used a complex natural local MI microenvironment to deliver and assemble scaffolds, thus paving the pathway for other SAP molecules with such progelator approach for in vivo cardiac injection. Moreover, this strategy can be exploited to use in conjunction with numerous small therapeutic molecules such as drugs and short peptides. This can even have the potential for targeted delivery of large therapeutic molecules, such as growth factors, and cells by injection of their mixture with the progelators [Self-assembling peptides (SAPs) are synthetic multifaceted polymers that can be customised to develop bioactive scaffolds via biocompatible supramolecular interactions. Their composition includes a series of hydrophobic and hydrophilic amino acids that can be exploited for tailored structure organisation by adding multivalent ions. They can incorporate bioactive peptide motifs to the main oligopeptide core motif that forms hydrogel ,52. Thesgelators .Bioactive motif seryl-aspartyl-lysyl-proline (SDKP) has been proven to hold pro-angiogenic, anti-inflammatory, and anti-fibrotic properties in various studies ,54,55. F2+ are added to a solution of sodium alginate, mild gelation and crosslinking occur resulting in alginate hydrogels preparations. The disruption of ECM with high extracellular Ca2+ levels together with apoptosis are the major events in large MI. The combination of alginate solution with calcium gluconate also known as a bioabsorbable cardiac matrix (BCM) is a novel strategy to evade pathological remodelling as it provides transient structural support to infarcted areas till the development of mature fibrotic tissue [2+ presence in the infarcted zone enables cross-linking of BCM into elastic ECM like scaffolding material that forms support for cardiac tissue, and it degrades gradually with time and ultimately is eliminated through renal excretion. Alginate hydrogel structures mimic ECM of living tissues that make it very suitable for numerous biomedical uses that involve regenerative tissue engineering, bioactive molecules delivery, cell implantation and wound healing. Alginate hydrogels are minimally invasive and also enable the controlled release of biomolecules such as drugs and proteins [Besides scaffolds constructed from synthetic polymers, several natural scaffolds have been developed. Alginate is an example of a natural anionic polymer commonly obtained from brown seaweed. In the biomedical field, it has been investigated extensively owing to its biocompatibility, low toxicity and low cost. Moreover, when divalent cations such as Cac tissue . Ca2+ prproteins . These pproteins ,60. A stproteins .Relevance of alginate hydrogels has also been demonstrated in clinical scenarios where it has proven to be a candidate for significant improvement of cardiac function in patients with cardiopathies. However, in these first studies, a short follow up time was applied ,63. MoreHyaluronic acid (HA) is another natural polymer that is anionic, non-sulfated glycosaminoglycan, highly hydrophobic and immunologically inert. It has significant structural and functional roles in ECM architecture, cell signalling and angiogenesis. Its use as a biomaterial has gained wide acceptance in tissue engineering due to its biocompatible, biodegradable and chemical modifiability features . Its useA study reported a viscoelastic adhesive patch termed as gel-point adhesive patch (GPAP) composed of ionically cross-linked and transparent starch hydrogel . This paSilk fibroin (SF) is an FDA-approved natural biomedical material that can be easily modified and possess very good mechanical stability and biocompatibility . A studyAnother FDA-approved natural biomaterial is chitosan (CS) hydrogel which has been widely used in tissue engineering, drug delivery and bone, cartilage, skin and nerve repair . The degOther studies have reported hydrogel scaffolds prepared from various biocompatible polymeric materials that included N-isopropylacrylamide (NIPAAm), poly N-isopropylacrylamide (PNIPAAm), 2-hydroxyethyl methacrylate (HEMA), methacrylate polylactide (MAPLA), dextran (Dex), poly(ε-caprolactone) (PCL) and gelatine ,80,81. PIrregular electric conduction is a common after-effect of MI. The physiological changes after MI include the replacement of necrotic CMs by collagen tissues and fibroblasts. This excess in ECM deposition by fibroblasts causes non-uniform electrical conductivity and CM uncoupling, thus inducing CMs vulnerable to re-entry. This mechanism favours ventricular arrhythmias to occur, thus leading to an increased risk of sudden cardiac death ,83. In aA very recent report has shown that cardiac scaffold based on electrospun silk fibroin (rGO/silk) coupled with reduced graphene oxide (rGO) can be optimised to present improved saturability and anisotropic conductivity in normalising post-MI cardiac functions. Four weeks after MI in the rat model, scaffold capability to improve CMs survival, lessen arrhythmias, thicken LV wall and enhance pumping function in addition to prominent effects on angiogenesis in infarcted myocardium was highlighted. Moreover, this study also evidenced that isotropic conductive rGO/silk patches and nonconductive silk ones did not show promising results in recovering the electrical environment, thus emphasising that optimised anisotropic scaffold composition is essential to achieve desired therapeutic function . AnotherCoadministration of various strategies is appreciated as it produces promising synergistic therapeutic results. A study has combined the application of a self-adhesive conductive hydrogel patch and injectable hydrogel in an infarcted region. Hydrogel patch constituted a homogenous network of dopamine-gelatine (GelDA) conjugates and dopamine-functionalised polypyrrole (DA-Py). The injectable hydrogel formulated in situ involved the Schiff base reaction between oxidised sodium hyaluronic acid (HA-CHO) and hydrazided hyaluronic acid (HHA). The co-administration of two systems in the infarcted region, HA-CHO/HHA hydrogel intramyocardially with a subsequent painting of conductive GelDA/DA-Py hydrogel patch on the cardiac surface, could outturn promising improvement of signal propagation in addition to enhancement of angiogenesis and cardiac function .2+ signal conduction and beating frequency. These effects also account for the synchronisation of CMs thus improving LV function [3+ triggered polymerisation in situ) hydrogels when painted in an infarcted rat heart surface-bound strongly and boosted electrophysiological signal conduction thus reviving cardiac function [Conductive poly-pyrrole-chitosan hydrogel (PPY-CHI)-based scaffold in preclinical MI animal model could produce considerably improved electrical conduction and synchronised cardiac contraction. In particular, narrowed QRS and QT intervals along with reduced fibrotic scar resistivity demonstrated more efficient conduction in post-MI hearts leading to a lower incidence of arrhythmias. The authors suggested that conductive polymers caused a reduction in arrhythmic events by acting on conduction velocity and/or tissue impedance in the scar tissue . Similarfunction . Pyrrolefunction . ImproveRecently, many studies have focused on the implementation of acellular scaffolds via functionalisation with various compounds/strategies to improve their effectiveness adding new features, properties, abilities and functions in a controlled way .In light of the importance of the ECM signals in driving the repair/regeneration of the infarcted heart, a few studies exploited conjugation between polymeric scaffolds and ECM components. In a study, the influence of integrated ECM along with mechanical anisotropy on scaffold function was investigated in a rat model of chronic MI employing three microfibrous poly (ester carbonate urethane) urea (PECUU) scaffold types that included longitudinal group, orthogonal group and ECM group . LongituMost of the studies in cardiac engineering address remodelling time points soon following MI. This acute strategy has very low significant relevance as much remodelling in the clinical scenario takes place unnoticed over a longer time frame that leads towards progressive cardiac failure . A very Many studies on MI pointed out the signalling pathways and related molecular factors involved in cardioprotection .In a pre-clinical porcine model, a microstructured PLGA/gelatine 3D cardiac patch with cardioinductive properties functionalised with adenosine was tested in in vivo ischaemic heart. It was shown to suddenly induce activation of the pro-survival RISK pathway that limited the myocardial injury. In fact, after three months, higher preservation of the myofibrillar structure of CMs accompanied by decreasing inflammation and fibrosis was detected . These fIn a very recent investigation, another research group produced a scaffold coupled with nitric oxide (NO), another cardioprotective agent . Exogeno2 generator in the infarcted microenvironment to impede inflammation and relieve hypoxia. In the rat MI model, this hydrogel displayed a reduction in apoptosis and infarct area, inhibition of inflammation, stimulation of angiogenesis, and almost full recovery of cardiac function [It is noteworthy that the abundant production of reactive oxygen species (ROS) plays a critical role in LV remodelling after MI . Ding etfunction . Other rfunction . Bioglasfunction .Growth factors have gained attention in the treatment of cardiopathies due to their direct response to various cellular functions such as migration, proliferation and adhesion. It is noteworthy that tissue regeneration is an important healing step post-MI. In this context, activation of endogenous resident cells and mobilisation of progenitor cells to the infarcted site has gained strong recognition as it can trigger the neovascularisation process and endogenous regeneration. Indeed, one of the hostile hurdles in post-MI recovery is angiogenesis impedance in the infarcted area thus leading to hypoxia and cell death. In this paradigm, growth factors can contribute to vasculogenesis and cardiac repair to evade HF. Formerly, the delivery approach commonly used was to infuse growth factors into systemic circulation; however, a major concern it encountered was the limited myocardial uptake and short half-life . For thiThe insulin growth factor-1 (IGF-1) plays a relevant role in the recruitment of endogenous stem/progenitor cells towards injured regions and stimulates angiogenesis . SubstanGranulocyte colony-stimulating factor (GCSF) holds the characteristic to recruit stem cells from bone marrow and induce their differentiation towards the endothelial cells or CMs in contact with infarcted myocardium. This unique ability of GCSF fuelled the scientific interest to exploit its beneficial aspects in the treatment of various cardiovascular diseases . KeepingThe basic fibroblast growth factor (bFGF) is a strong growth factor that favours stem cell survival and induces neovascularisation ,113. bFGIn numerous studies, scaffolds coupled with vascular endothelial growth factor (VEGF) have been documented to prevent adverse LV remodelling and recover cardiac function in infarcted rodents. VEGF coupled with alginate-CS hydrogel  and Dex-Myeloid-derived growth factor (MYDGF), a paracrine-acting protein has been shown to recover cardiac function in infarcted hearts in pre-clinical studies ,123. YuaNeuregulin is an epidermal growth factor that acts as a signalling protein which bind to ErbB4 receptor expressed by CMs that results in activation of downstream signalling pathways which promote cardioprotective effects . Cohen aInterestingly, Kontonika et al. used alginate-based scaffolds to deliver growth hormones (GH) in a rat model in which ischemia-induced arrhythmogenesis was induced to study its effect on ventricular tachycardia and heart rate variability. During an observation period of 24 h, ventricular tachycardia and sympathetic activation were attenuated while infarct size was unaffected . HoweverSteele et al. fabricated a shear-thinning, self-healing, bioengineered hydrogel (SHIELD) composed of a copolymer of PEG vinyl sulfone (PEG-VS) and customised peptide P1 to couple a protein-engineered dimeric fragment of Hepatocyte growth factor (HGFdf). This hydrogel has demonstrated synergistic therapeutic outcomes of SHIELD and HGFdf on ischemic hearts in rat models to limit post-infarction adverse LV remodelling . RecentlOn the basis that heart-derived cardiac stromal cells (CSCs) hold regenerative therapeutic effects by secreting paracrine mediators and interacting with CMs ,132. HuaExtracellular vesicles (EVs) are membrane vesicles enclosing cytosol in the lipid bilayer that have originated from different subcellular compartments. EVs are heterogeneous populations secreted by cells and include exosomes and microvesicles . EVs havEVs secreted by MSCs possess cardioprotective effects thus displaying the potential to treat MI . FurtherIt should be kept in mind that EVs are enriched with a unique representative cargo of their origin cells. Therefore, the selection of cell types to derive and extract EVs is essential to determine the therapeutic window of EVs. It is noteworthy that iPS-CM-derived EVs are rich in cardiac-specific miRNAs that have the potential to modulate CM-specific processes. Actually, collagen hydrogel patch encapsulated with iPS-CM-derived EVs enables the extended release of EVs thus allowing direct and continuous treatment when implanted in infarcted rat hearts. Besides the other effects, in this case, the authors observed also the antiarrhythmic effect and reduced pathological hypertrophy .Many proteins are actively involved in tissue regeneration and repair. In this context, selective and specific modification of proteins or peptides can be employed to optimise biomaterial properties and biological effects.Histone deacetylases (HDACs) are an enzyme family that regulates gene transcription by deacetylating key lysine residues on histones and playing a crucial function in recruiting various epigenetic regulators of gene promoters/enhancers on DNA, hence modulating various aspects of metabolic anomalies including cardiovascular pathologies ,145. 7-aMcLaughlin and co-workers tailored injectable hydrogel matrices composed of recombinant human collagen type I (rHCI) and type III (rHCIII) that were crosslinked by N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and were glycosylated by glycosaminoglycan chondroitin sulphate C (CS). Interestingly, in situ assembly and cross-linking of these hydrogels enabled the formation of a biomimetic 3D matrix that promoted the polarisation of endogenous macrophages towards a pro-wound healing M2 phenotype, restored mechanical properties, reduced scar size, prevented heart enlargement and encouraged CMs survival to avert LV remodelling within infarcted myocardium in pre-clinical models. In comparison to rHCIII matrices, rHCI matrices were therapeutically more significant to avert LV remodelling when applied in the late proliferative phase post-MI .Another research group employed sericin, a silk-derived protein used as a wound-healing agent, for the first time. Genipin-crosslinked sericin hydrogel, injected in the heart infarcted zone of mouse MI model, decreased scar production, improved LV wall thickness, promoted vasculogenesis, impeded inflammatory responses and reduced apoptosis, thus inducing a significant improvement in cardiac function and averting LV remodelling .Recently, Feng et al. hypothesised that angiogenic peptides derived from VEGF have the potential to mimic the bioactivity of VEGF and can activate its receptor to promote angiogenesis. Their study tailored a scaffold composed of collagen and decellularised ECM to encapsulate angiogenic peptides . BesidesAfter MI, a cascade of structural and biological events begins within the infarcted region. Disequilibrium in the proteolytic pathways in the infarcted region, such as the imbalance between matrix metalloproteinases (MMPs) and endogenous tissue inhibitors of metalloproteinase (TIMPs), is one of the biological events that contribute towards adverse LV remodelling. MMPs influence the remodelling course by contributing to ECM turnover and inflammatory signalling to remove necrotic CMs ,154. At Within this therapeutic framework, targeted delivery strategies could be a logical and promising approach. In current bioengineering approaches, therapeutic scaffolds could hold promise to effectively target proteolytic pathways such as the MMP system. In this context, a pre-clinical research study was performed using an HA-based MMP-sensitive hydrogel (HAMMPS) with recombinant tissue inhibitor of MMPs encapsulated into it, to target the myocardial ECM proteolytic system in large animal models. In this study, full-length recombinant protein (rTIMP-3) was incorporated into the hydrogel that encompasses MMP-cleavable peptide cross-links to control the rTIMP-3 release from the hydrogel. After 28 days, in the swine MI model, the implant of scaffold incorporating rTIMP-3 (HAMMPS/rTIMP-3) showed promising outcomes that included a 50% decrease in LV dilation and extent of MI wall thinning, attenuation in indexes of HF such as left atrial size and LV filling pressures as well as a significant decrease in transcriptional profile for myofibroblasts together with profibrotic pathways. In brief, the results highlighted that localised therapeutic delivery of MMP-sensitive scaffolds that release rTIMP to target myocardial matrix can improve LV function, architecture and remodelling after MI . The effIn other studies, this approach was also combined with growth factors/chemokines release. Awada and collaborators designed a composite hydrogel composed of fibrin gel and heparin-based coacervates loaded with TIMP-3, FGF-2 and SDF-1α . In vivoFurther broad investigations in pre-clinical studies could elucidate in more detail the therapeutic influences of TIMP in the infarcted zone. To date this strategy has been applied at injectable scaffold only; further study in which this new approach if extended to patches could be a promising tool for cardiac remodelling treatment.Commonly the administration of the drugs in cardiac patients is done through a simple systemic delivery approach, which in general results in low efficiency, drug toxicity along off-target side effects . ScaffolColchicine (Col) is a natural anti-inflammatory drug that is used limitedly owing to systemic toxicity . A studyIt is noteworthy that some drugs exert cardioprotective effects, such as Salvianolic acid B (SaB) , howeverAnother study documented a nanodrug-loaded ROS sensitive hydrogel system composed of hyperbranched poly(β-amino esters) (HB-PBAEs), polydopamine deposited tanshinone IIA NPs (TIIA@PDA) and thiolate modified HA (HA-SH). HB-PBAEs and TIIA@PDA crosslinks with HA-SH when simultaneously injected in the infarcted heart, thus resulting in in situ hydrogel formation. This hydrogel in infarcted rat MI has been shown to improve ventricular function, increase LV wall thickness as well as reduce infarct size and inflammation . RecentlMethylprednisolone (MP) is a glucocorticoid drug that exhibits the potential to promote vascularisation and reduce inflammation, myocardial injury and collagen content in MI experimental models . In thisGene therapy is an attractive therapeutic strategy to counteract HF. However, its efficiency, safety and targeted delivery are still a matter of concern . Among pGu and collaborators employed scaffolds made of elastomeric polyester urethane urea (PEUU) and polyester ether urethane urea (PEEUU)-based patches loaded with AAV encoding reporter gene green fluorescent protein (GFP) to assess the transduction efficiency in rodents three days post-MI. Follow-up at 12 weeks evidenced that GFP protein expression could be observed in α-SMA- and cardiac troponin T-positive cells, thus suggesting targeted delivery of AAVs. Comprehensively, the study emphasised viral transduction and prolonged release of genes that, along with elastomeric mechanical support, provided evidence for the scaffold coupled with AAVs as a suitable therapeutic strategy for improvement of cardiac function post-MI. Specific genes of interest might be incorporated in AAVs in future work and their release kinetics tailored by coupling AAVs with scaffolds to target infarcted regions . HoweverOn the other hand, miRs are an important evolutionary element to post-transcriptionally regulate gene expression by silencing mRNA that inhibits their translation and leads them towards degradation . In respAs miR-1825 acts as a key regulator to increase expression of miR-199a, a strong miR that promotes cardiac regeneration in rodents, Pandey and collaborators designed gelatine and silicate-based hydrogel loaded with AAV-miR-1825 and tested its therapeutic efficacy in the mice MI model . Direct In cardiac tissues, miR-29B is linked to the regulation of fibrosis and ECM remodelling due to its considerable role in collagen production . It is wBased on the critical role of angiogenesis in the post-MI heart, some authors applied gene therapy to stimulate it, exploiting the miR-17-92 cluster . miR-92aThese studies based on miRNA-based scaffolds as a therapeutic strategy for MI have highlighted that targeting miRNA have a high potential to treat ischemia and modulate different aspects of LV remodelling. It appears to be relevant to design further studies with longer follow-up windows to ascertain the maintenance of the relevant gene expression control.Acellular scaffolds are an attractive approach that has gained extensive attention in regenerative cardiac medicine, with a specific interest in the limitation of adverse LV remodelling. The major advantages of acellular scaffolds are immediate implantability and customisable morphological design for stimulation of tissue regeneration, providing integration and tuned mechanical support at infarcted sites. In addition, acellular scaffolds can be easily produced, transported and stored, and for these reasons, there is a strong interest from the biomedical companies for this solution.Ideally, an acellular cardiac patch has to contain the necessary regulatory signals to stimulate precise reactions at a molecular level to direct cell behaviour in terms of stem cell recruitment, cardiac and/or endothelial differentiation and electrical coupling between adjacent cells to recapitulate developmental processes in morphogenesis. A relevant future direction is to further impinge on the ability of the engineered scaffold to crosstalk with resident cells in a precise and controlled manner through spatial and temporal control as this is a critical requirement. Biodegradable polymeric materials are particularly suitable to be processed using different microfabrication techniques to obtain micro-nano topography mimicking the anisotropic structure of the myocardium providing a spatial interface to control adhesion, elongation, ordered disposition and myocardial commitment of stem cells.Other emerging directions are the concept of open scaffold platforms that can be adapted to the patients’ needs and a better search for mininvasivity. Acellular natural and synthetic polymeric scaffolds can be combined with drugs, specific molecular signals or nanosystems to achieve cardiac tissue repair and sustain cardiac function in the long term . RecentlA well-reasoned choice of relevant pre-clinical and clinical models is another future challenge. The research in this field is still far limited to pre-clinical models and most of the studies consisted of implant or injection of scaffolds shortly after MI induction, conditions difficult to reproduce in clinical scenarios. A deeper knowledge about the timing of intervention is needed, as for the medical treatments also the right time of surgery plays a key role in the improvement of post-infarct patient outcomes. For this reason, more studies to be performed in vivo chronic models are required. The experiments should be performed also when the cardiac remodelling has already developed, thus mimicking the timing of intervention in the post-MI patients by the clinicians/surgeons. Further, extensive research is still required for monitoring the fate of implanted scaffolds in pre-clinical studies to evaluate their long-term impact on cardiac function. For this reason, a joint effort should be made by both researchers and clinicians to achieve suitable and well-designed experimental protocols that could meet surgical requirements, to obtain fundamental knowledge that may allow one to transfer this biomaterial-based strategy from bench to bedside.After several years of investigation in this field, it can be concluded that besides mimicking myocardial properties, the acellular scaffold should be functionalised with several strategies to achieve the multiple desired benefits, including a reduction of scar extension, formation of new vessels, as well as an improvement in cardiac function and electrical conductivity. The results of the reported studies highlighted that the specific properties of the scaffolds can impact different aspects of cardiac remodelling, thus suggesting that only combinatorial approaches will allow synergistic beneficial effects. The current knowledge and progress on acellular scaffolds will pave the way to plan effective approaches for the construction of clinically operative acellular scaffolds and direct the research towards humans to assess their clinical success.Remarkable preclinical advances have been made for the use of acellular scaffolds alone or in combination with biotherapeutic moieties to limit LV remodelling post-MI. This compelling progress in the field of biomaterials and cardiac engineering of therapeutic acellular scaffolds can be considered a very attractive option for MI treatment in the clinical setup in future. Further in-depth investigation on the mechanism of action of acellular scaffolds will provide further novel cues for the customised engineering of these acellular scaffolds. Although it is just the beginning of this therapeutic approach, the large quantum of efforts made till now has provided noteworthy clues to rapidly accelerate further developments.


T cells must recognize pathogen-derived peptides bound to major histocompatibility complexes (MHCs) in order to initiate a cell-mediated immune response against an infection, or to support the development of high-affinity antibody responses. Identifying antigens presented on MHCs by infected cells and professional antigen-presenting cells (APCs) during infection may therefore provide a route toward developing new vaccines. Peptides bound to MHCs can be identified at whole-proteome scale using mass spectrometry—a technique referred to as “immunopeptidomics.” This technique has emerged as a powerful tool for identifying potential vaccine targets in the context of many infectious diseases. In this review, we discuss the contributions immunopeptidomic studies have made to understanding antigen presentation and T cell priming in the context of infection and the potential for immunopeptidomics to inform the development of vaccines to address pressing global health problems in infectious disease. Recognition of pathogen-specific peptide-major histocompatibility complexes (pMHCs) is required for naive T cells to become activated and proliferate, and for effector T cells to recognize infected cells and carry out effector functions such as cytokine secretion and lysis of infected cells that ultimately lead to control of an infection. Antigen-specific T cell responses are also required for B cells to undergo affinity maturation and produce high-affinity antibodies . Identif+ and CD4+ T cells, respectively, and are loaded with peptides via distinct pathways of antigen processing that have been reviewed in detail elsewhere  provides a means of experimentally identifying the repertoire of peptide antigens presented on MHCs by cells infected with a pathogen of interest. In a typical immunopeptidomic workflow, MHC-peptide complexes are immunoprecipitated (IP) from a population of cells of interest using an HLA-allele-specific antibody or pan-HLA antibody. Peptides are released by acid elution, MHCs are separated from peptides by size exclusion or solid-phase extraction, and eluted peptides are analyzed by liquid chromatography coupled to tandem MS (LC-MS/MS)  5, 6). , 6. 5, 6MS analyses of MHC peptides can be carried out in two distinct modes. In data-dependent mode, peptides are detected and prioritized for tandem mass spectrum acquisition based on their abundance, without prior knowledge of the composition of the sample, enabling discovery of novel vaccine targets. In targeted mode, a predetermined list of targets is selected for acquisition, enabling studies of the mechanism and kinetics of presentation of known antigens by providing reliable detection and quantification across multiple samples.Here, we review the use of MS-based immunopeptidomics to study antigen presentation in infectious disease. These studies reveal basic insights into the biology of antigen presentation, provide data sets for training predictive models of antigen presentation, and guide the rational selection of vaccine targets. We go on to discuss opportunities for further study in this area, technical pitfalls that remain to be overcome, and the potential for immunopeptidomics to inform vaccine development.Vaccinia virus infection has served as a model system for research on the basic biology of antigen presentation in viral infection. Vaccinia virus is a large, enveloped poxvirus and a relative of variola virus . It was widely used as a vaccine against smallpox before the eradication of the disease, and some variants such as Modified Vaccinia Ankara (MVA) are being used experimentally as viral vectors for delivery of vaccine antigens –9. Altho–An immunopeptidomic study of vaccinia virus-infected cells assessed whether most viral peptides presented by MHCs are immunogenic or if a large proportion are immunologically silent . The autVaccinia virus infection has also been used as a model to study the kinetics of antigen presentation during viral infection and the relationship between pMHC abundance and immunogenicity. Croft et al.  used a tin vitro binding assays  vaccine strain  generally requires combining immunopeptidomics with techniques that can directly measure a T cell response against a given peptide of interest. Traditionally, this has been done by measuring production of IFN-γ by T cells specific for each epitope of interest . T cells77–Mycobacterium tuberculosis can be presented by both infected cells and uninfected bystander cells (Cell-to-cell heterogeneity in antigen presentation (problem 2) may be consequential for immunity to some infections. For example, certain antigens of er cells , 81, whier cells , 83 can er cells , 85, pro6 to 107 cells (8 to 109 cells (problem 3) is often required to detect pathogen-derived peptides. The use of microfluidic devices or other low-volume sample handling techniques .Whereas recent immunopeptidomic studies in cancer immunology have used sample input on the order of 10chniques  could hechniques . Lower sCombining computational modeling with targeted mass spectrometry approaches could provide another avenue toward reducing sample input requirements (problem 3). Targeted MS analyses can detect MHC-associated peptides with greater sensitivity than data-dependent analyses, potentially reducing sample input requirements and increasing the likelihood of detecting peptides that would not be detectable in a data-dependent analysis. Accurate predictive models of peptide presentation on MHCs could identify promising candidates to experimentally validate using highly sensitive targeted MS workflows, and these experimental results could in turn be used for further model refinement.Future improvements in immunopeptidomic methods may also focus on overcoming technical pitfalls that can result in inaccurate identification of MHC peptides by MS. In one instance, for example, HIV-derived peptides previously identified as MHC class I ligands  were sho88–Immunopeptidomics has already been used to design vaccines that improve control of infection in mouse models of chlamydia , leishma


Faced with the challenges posed by infectious diseases and cancer, nucleic acid vaccines present excellent prospects in clinical applications. Compared with traditional vaccines, nucleic acid vaccines have the characteristics of high efficiency and low cost. Therefore, nucleic acid vaccines have potential advantages in disease prevention and treatment. However, the low immunogenicity and instability of nucleic acid vaccines have limited their development. Therefore, a large number of studies have been conducted to improve their immunogenicity and stability by improving delivery methods, thereby supporting progress and development for clinical applications. This article mainly reviews the advantages, disadvantages, mechanisms, delivery methods, and clinical applications of nucleic acid vaccines. Since the first vaccines were developed over 200 years ago, vaccines have directly decreased the morbidity and mortality caused by dangerous diseases across large human populations . Furtherin vitro transcribed (IVT) mRNA in animals began in 1990. At that time, the gene of encoding the mRNA sequence was injected into mice, and then researchers detected the produced protein  and RNA [as messenger RNA (mRNA)] vaccines, exhibit promising potential in targeting various indications and diseases. Furthermore, cancer vaccines present an attractive strategy that can induce specific and persistent immune responses against tumor antigens. DNA vaccines are based on bacterial plasmids that encode antigens and immunostimulatory molecules . The first case of DNA vaccine-mediated immunity began in the 1990s, when plasmid DNA encoding the influenza A nucleoprotein led to a protective and specific cytotoxic T lymphocyte (CTL) response . Additio protein .In this review, we first provide an overview of current understanding of the nucleic acid vaccines. We next focused on the delivery of nucleic acid vaccines, with an emphasis on improving efficacy. Finally, we turned our attention to the clinical applications of nucleic acid vaccines by elaborating on the prospects and needed improvements.DNA vaccines are generated by inserting a gene encoding antigens into a bacteria-derived plasmid, which needs to be controlled by a powerful promoter . DNA plDNA vaccines can induce both humoural and cellular immune responses, and DNA can be delivered through a variety of routes, including intramuscular (IM), intradermal (ID), mucosal, and transdermal delivery. For DNA vaccines, after internalization, the DNA is transferred to the nucleus for transcription and then translated in the cytoplasm .+ T cells through major histocompatibility complex (MHC) class I complexes on the somatic cells; (2) antigen presentation relies on professional antigen-presenting cells (APCs), for instance, dendritic cells (DCs), that are transfected by the plasmid DNA at the injection site, and then present the expressed antigens to T cells through MHC class I and II complexes; and (3) professional APCs phagocytose plasmid-transfected somatic cells, resulting in cross-priming and presentation of antigens to both CD4+ and CD8+ T cells. At muscle sites, since the presentation of antigens through MHC class II is needed to induce CD4+ helper T cells, cross-presentation is probably the major route, possibly through processing of apoptotic cell debris  aftl debris . Even inl debris .+ and CD8+ T cells and indirectly activating humoural immunity. Thus, DNA vaccines not only retain many encoded safety features but also retain the specificity of subunit vaccines. Third, safety is another advantage of DNA vaccines. Prior research has clearly demonstrated the safety of DNA vaccines in animal models and human clinical trials  vaccine induced anti-DNA antibody, and triggered autoimmune syndromes while combination with required adjuvants . In cont7Gp3N(N: any nucleotide)], which is a 7-methyl-guanosine residue (m7G) bound to the 5′-end of the RNA transcript via a 5′–5′ triphosphate bond with any nucleotide; a 5′ untranslated region (5′UTR) that sits immediately upstream of the translation initiation codon; an open reading frame (ORF) encoding the gene of interest (GOI); a 3′ untranslated region (3′UTR); and a tail of 100–250 adenosine residues [poly(A) tail]  tail]   tail] . Among tA) tail] . SimilarA) tail] , while tA) tail] . In compA) tail] .in vivo. When antigen-encoding mRNAs are delivered into APCs in efficient ways , the mRNAs can be released and translated into relative antigenic proteins in the cytoplasm. Then, they are processed into peptide epitopes, which are subsequently combined with the MHC class I via a cross-presentation pathway. In this part, the activation of CD8+ T cells results from the transfer of MHC-peptides complexes to the cell surface of APCs, which leads to a corresponding immune response  RNase-mediated degradation, (2) high molecular weight, and (3) electrostatic repulsion resulting from the interaction between the negatively-charged mRNA molecules and the negative charge of the proteoglycan-coated cell membrane , which lin vivo is essential to achieve therapeutic goals. Nucleic acid vaccines have to enter the nucleus or cytoplasm, where transcription or protein expression, respectively, can take place.Effective DNA or mRNA delivery As previously mentioned, the greatest challenge of nucleic acid vaccination is low immunogenicity due to cross-membrane barriers. DNA vaccines need to penetrate the nuclear membrane barrier for delivery enhancement, and mRNA vaccines have to cross the lipid-based plasma membrane as efficiently as possible. Hence, several methods to enhance cell delivery and immunogenicity have been developed.in vivo electroporation for mRNA delivery can induce a strong immune response in mice due to increased mRNA release into the cytoplasm in the context of non-amplifying mRNA or self-amplifying mRNA administration , intradermal (ID), mucosal, and transdermal delivery. The failure of nucleic acid vaccines to elicit a strong immune response in humans is due to needle injection delivery. Currently, physical delivery methods using a gene gun or intradermal electroporation may facilitate transportation and enhance immunogenicity , 2015. Pstration . In addiectively .The efficacy of nucleic acid vaccines can be improved significantly by utilizing chemical delivery methods . Currently, the components of nanocarriers used in nucleic acid vaccines can be classified into lipid-based nanosystems , polymerToxoplasma gondii DNA vaccine with an alum adjuvant can improve the survival rate of mice  for “mRNA vaccine for cancer prevention” clinical trials, a total of 92 results In response to a wide variety of infectious diseases that harm humans, a certain number of DNA vaccines have been developed and have entered the clinical trial stage. However, because some fatal shortcomings have not been resolved, most of the DNA vaccines officially approved for use in the market are aimed at animals for veterinary treatment. For instance, it has been reported that a canarypox vaccination used to treat West Nile virus infection can produce an effective immune response in horses and dogs . HoweverThe viral diseases that are of relatively great concern in clinical trials mainly include AIDS, hepatitis B and C, influenza and warts or cancer caused by human papillomavirus (HPV). Existing research institutions have designed a DNA vaccine for HSV-2, and found that if the DNA vaccine is combined with the adjuvant Vaxfectin , it can Plasmodium falciparum, a clinical trial evaluated different anti-malarial DNA vaccines. In addition, the European research project LEISHDNAVAX was designed to create and test an effective DNA vaccine against leishmaniasis  Dendritic cell mRNA vaccines.Infectious disease vaccines developed via DC cells are mainly limited to therapeutic vaccines against HIV-1. A clinical trial in humans evaluated CMV pp65 mRNA-loaded DC vaccination of healthy human volunteers and administration of allogeneic stem cell receptors, and the induction and expansion of the CMV-specific cellular immune response were found .(2) Direct injection of non-amplifying mRNA vaccines.In an animal trial, researchers used the protamine-based RNActive platform that encodes the rabies virus glycoprotein for immunization. The results showed that in mice, the vaccine was able to induce protective immunity against rabies virus, which can cause fatal inflammation in the brain of mice and produce an effective neutralizing antibody response in pigs . Other i+ T cell response  glycoprotein or S protein which serves as the main trigger of neutralizing antibody, such as full-length S protein or S1 receptor binding domain (RBD) and expressing in virus-like particles (VLP), DNA or viral vector . ModernaOne of the earliest clinical trials on cancer treatment using DNA vaccines was conducted in 1998 . This tr+ T cell immune response. A phase I clinical trial evaluating the Mam-A cDNA vaccine  of several patients increased. These results illustrated the safety of the DNA vaccine and suggested that it might have an impact on the tumor growth rate (The Mammaglobin-A (Mam-A) cDNA vaccine can induce antitumour- immunity in breast cancer. The vaccine encoding Mam-A cDNA can generate a Mam-A-specific CD8 vaccine  observedwth rate .H1)-biased cellular immune response. In the different dose groups, the average peak value of the T cell response induced by VGX-3100 was 642 to 1458 SFU per 106 PBMCs. The results showed that the vaccine induced strong and extensive cellular immunity. In addition, no serious adverse reactions, grade 3/4 adverse reactions or injection site reactions were observed, which showed the safety of VGX-3100  16 and 18 E6/E7 antigens, was tested. This trial evaluated 18 postresection patients with grade 2/3 cervical intraepithelial neoplasia (CIN) who received a three-dose regimen  via three intramuscular injections. An IFN-γ ELISpot assay found that 78% of subjects had an increased T helper 1  and non-traditional  routes. Several studies have shown that naked mRNA injected intranasally can be selectively absorbed by DCs, thereby causing an effective prophylactic or therapeutic antitumor T cell response . In prioPeople have long been concerned about the safety of DNA vaccines, mainly due to the stable integration of transfected DNA into the genome of somatic or germinal cells, which may lead to dysregulation of gene expression and gene mutation. The overall safety of DNA vaccine has been fully demonstrated in several clinical trials, and adverse reactions are limited to local reactions at the injection site . TherefoAlthough clinical trials using DNA vaccines have found that these vaccines can effectively induce cellular and humoural responses, the strength of these reactions is usually not sufficient to produce significant clinical benefit. Additionally, due to tumor immune tolerance to endogenous autoantigens, DNA vaccines still need to be improved in regard to inducing effective antigen-specific cellular immune responses. Therefore, the development of a method to bypass immune tolerance is warranted in DNA vaccine development. In addition, DNA vaccines can be used in combination with other cancer treatments to further fight against tumors .Because there are no toxic chemicals involved in the production of mRNA, and the environment of the platform is clean and free of foreign virus contamination, the production risk of mRNA is significantly lower than that of other vaccine platforms . After vaccination, the theoretical risk of infection and integration of the vector into host cells will not exist, unlike in DNA vaccination . In shorCurrently, nucleic acid vaccines are undergoing rapid development for the treatment of infectious diseases and cancers. Pandemics and malignant tumors, such as HIV, AIDS, Ebola, COVID-19, breast cancer, and melanoma, have raised people’s awareness of the global threats to human health and promoted the development of nucleic acid vaccine platforms, enabling researchers to cope with the challenges of severe situations. The dozens of preclinical and clinical studies indicate that nucleic acid vaccines not only have a significant efficacy in the treatment of infectious diseases, but also have demonstrated potency in cancer applications. Although nucleic acid vaccines provide a certain number of advantages over conventional vaccines, further optimization is necessary before they become the main treatment strategy for human patients. This article summarizes the optimization strategies based on the shortcomings of nucleic acid vaccines from three perspectives: physical methods , chemical methods , and adjuvants. Therefore, the uptake and membrane-penetrating capacity of nucleic acid vaccines can be promoted, resulting in a stronger immune response. An increasing number of molecular adjuvants, such as adhesion molecules, cytokines, chemokines, and transcription factors, are undergoing safety and tolerability assessment. Similarly, the continuous development of vaccine delivery methods is promising and worthy of further research. While it seems unlikely that a technology that can provide a suitable solution for every single patient will be developed, combining existing technology and constantly improving the understanding of human immunology will provide better tools to fight known and emerging global threats.All authors read and approved the final manuscript. FQ wrote the initial manuscript. FX contributed new ideas and created the figures. HC and BC created 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.


Tumor recurrence still represents a major clinical challenge for cancer patients. Cancer cells may undergo a dormant state for long times before re-emerging. Both intracellular- and extracellular-driven pathways are involved in maintaining the dormant state and the subsequent awakening, with a mechanism that is still mostly unknown. In this scenario, cancer metabolism is emerging as a critical driver of tumor progression and dissemination and have gained increasing attention in cancer research. This review focuses on the metabolic adaptations characterizing the dormant phenotype and supporting tumor re-growth. Deciphering the metabolic adaptation sustaining tumor dormancy may pave the way for novel therapeutic approaches to prevent tumor recurrence based on combined metabolic drugs.Tumor relapse represents one of the main obstacles to cancer treatment. Many patients experience cancer relapse even decades from the primary tumor eradication, developing more aggressive and metastatic disease. This phenomenon is associated with the emergence of dormant cancer cells, characterized by cell cycle arrest and largely insensitive to conventional anti-cancer therapies. These rare and elusive cells may regain proliferative abilities upon the induction of cell-intrinsic and extrinsic factors, thus fueling tumor re-growth and metastasis formation. The molecular mechanisms underlying the maintenance of resistant dormant cells and their awakening are intriguing but, currently, still largely unknown. However, increasing evidence recently underlined a strong dependency of cell cycle progression to metabolic adaptations of cancer cells. Even if dormant cells are frequently characterized by a general metabolic slowdown and an increased ability to cope with oxidative stress, different factors, such as extracellular matrix composition, stromal cells influence, and nutrient availability, may dictate specific changes in dormant cells, finally resulting in tumor relapse. The main topic of this review is deciphering the role of the metabolic pathways involved in tumor cells dormancy to provide new strategies for selectively targeting these cells to prevent fatal recurrence and maximize therapeutic benefit. Tumor dormancy is defined as a temporary, reversible mitotic and growth arrest  physioloDormancy represents a basic survival strategy providing a selective advantage for organisms to persist in a hostile environment. Tumor cell dormancy, therefore, exploits evolutionarily conserved mechanisms of adaptation to succeed during stressful steps in tumor progression . From thIn the early phases of tumorigenesis, dormant cells may accumulate mutations in a dynamic multistep process characterized by a hyper-proliferative reactivation followed by a period of dormancy from which cancer cells may exit to undergo a final malignant transformation, lastly resulting in tumor mass formation .During the multistep process of metastasis formation, a small fraction of “disseminating tumor cells” (DTCs) invade surrounding tissues, intravasate, survive the circulation, extravasate, and finally colonize the secondary organ, where they need to adapt to the new non-permissive microenvironment and re-start proliferating, ultimately resulting in a second metastatic lesion . MetastaBesides its role in quiescent cells, in the primary tumor and the arising metastatic lesion, dormancy is also a key feature of cancer cells surviving the treatment with anti-proliferative chemotherapeutics drugs. These cells, also termed “drug-tolerant persister cells” (DTPCs), can be considered as a “minimal residual disease”, able to escape the selective pressure of anti-cancer drugs by entering into a dormant condition . It is sIn summary, the ability to undertake a period of dormancy represent a necessary adaptation for cancer cells to support metastatic dissemination and tumor relapse following therapy. Therefore, understanding the molecular basis and the metabolic strategies of cancer cells undergoing dormancy is essential for developing efficacious approaches to prevent tumor relapse.Cancer cell dormancy prevents cell death under stressful conditions by inducing a G0 cell cycle arrest which results from both the alteration of intrinsic and autocrine signaling, and the occurrence of external-inducing inputs derived from the microenvironment .Several alterations in genes associated with proliferation and differentiation correlate with the initiation and maintenance of the dormant phenotype. For instance, the overexpression of the FBXW7 gene, encoding a component of specific E3 ubiquitin ligase, leading to the degradation of the positive cell-cycle regulators cyclin-E and c-Myc, is essential for the maintenance of dormancy in breast cancer DTCs . SimilarMoreover, the phenotypic plasticity characterizing cellular dormancy is mostly governed by dynamic epigenetic mechanisms ,38. In oImportantly, cancer cell dormancy can also be controlled by alterations in the main survival/cell cycle signaling pathways. In particular, the ERK/p38α balance plays a central role in modulating the dormant phenotype by inducing and integrating different signaling pathways in cancer cells . SpecifiFinally, the absence of proliferation-inducing stimuli may also lead to dormancy. In pancreatic cancer, the ablation of the oncogenic drivers mutant KRAS and c-MYC induces a dormant state, allowing the survival of a few residual cancer cells that rely on autocrine IGF1/AKT as an adaptive mechanism . PancreaTogether, the induction of these intrinsic pathways results in the activation of response to stress strategies such as autophagy  and the Coherently, dormancy correlates with the transcriptional up-regulation of driving autophagy-related genes, such as LC3, ATG4, ATG5, ATG7, and BECN1 .Besides removing misfolded proteins, the regulation of mitochondrial functionality and the prevention of ROS accumulation are of extreme importance for the long-term survival of dormant cells. To this aim, dormancy is frequently associated with increased mitophagy as a major mechanism to eliminate damaged/dysfunctional mitochondria to manage oxidative stress balance ,72. GiveThe UPR is a multifaceted network of signal transduction pathways activated when the endoplasmic reticulum (ER) is functionally altered. UPR is triggered by the activation of three primary stress sensors: inositol-requiring protein 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6) . These sMoreover, the ERK/p38α balance plays a central role in modulating the dormant phenotype by inducing and integrating different signaling pathways in cancer cells . In partIn addition to intrinsic pathways, cancer cells might enter the dormant state as a response to external signals from the tumor microenvironment (TME). The dynamic interaction between cancer cells and the TME plays a crucial role in modulating dormancy entry/escape . This asCancer-associated fibroblasts (CAFs) may influence cancer dormancy by producing and secreting a collection of different molecules, including TGFβ, interferons, interleukins, chemokines, and other cytokines . In ER-bImportantly, the composition of the immune population in the host tissue also plays a driving role in the modulation of the dormant phenotype in tumor cells. In a murine sarcoma model, an increased proportion of anti-tumor immune cells, especially natural killer cells (NKCs), compared to immunosuppressive cells, has been associated with the maintenance of tumor dormancy . Indeed,Besides cellular components, the extracellular matrix (ECM) composition may also control the dormant status in cancer cells. Loss of integrin activation and fibronectin matrix re-organization are two driving processes that maintain dormancy by contributing to p38 activation and ERK inhibition ,92. ImpoAmong extracellular stimuli, hypoxia has been identified as a pivotal contributor to tumor dormancy . Indeed,The accumulation of HIF-1α under normoxic conditions, a phenomenon known as “pseudohypoxia” , may alsFinally, the absence of proliferation-inducing stimuli may also lead to dormancy. In pancreatic cancer, the ablation of the oncogenic drivers mutant KRAS and c-MYC induces a dormant state, allowing the survival of a few residual cancer cells, which rely on autocrine IGF1/AKT as an adaptive mechanism . PancreaIn conclusion, several factors may influence tumor cell entry in a dormant state, making this adaptive strategy particularly complex and multifaceted, but providing numerous possible strategies to prevent dormancy-related tumor relapse and metastatic disease.One of the most intriguing aspects of dormant cells is their ability to interpret homoeostatic signals from the microenvironment. This feature allows cancer cells to evade immune surveillance and chemotherapy during the dormant phase and, on the other side, to reawaken in response to specific signals, re-acquiring proliferative and metastatic potential and finally re-establishing detectable tumor masses. However, despite the growing interest in these issues in recent years, stimuli inducing dormant cells awakening are still largely unknown, and further studies are needed to understand the molecular mechanism driving these phenomena.Remodeling microenvironmental composition is one of the main drivers of cancer cell dormancy escape. Indeed, the transition from dormancy toward a proliferating state relies on the ability of tumor cells to adhere to ECM as demonstrated by the induction of dormancy escape following deposition of type I collagen at the metastatic location, promoting the reawakening of disseminated DCTs by inducing cytoskeletal reorganization and β1/β4-integrin signaling  or the fAnother leading factor of dormancy’s escape is the presence of new vessels that may reactivate tumor growth in a process known as “angiogenic switch”. Indeed, it is well known that anti-angiogenic therapies may induce tumor dormancy, while angiogenic ones promote tumor rescue . The proChanges in the immune system composition, especially at the tumor immune microenvironment level, such as during ageing or following the treatment with chemotherapy and immunosuppressive drugs, may drive the escape from dormancy . MoreoveUnderstating signals and molecular mechanisms driving the escape of cancer cells from the dormant state is relevant for developing therapeutic approaches specifically targeting dormant cells, making them again sensitive to anti-proliferative drugs.A strict connection exists between metabolism and cell cycle progression. Proliferating cells display higher biosynthetic requirements than non-proliferating cells to double in size and synthesize proteins, lipids, and DNA to generate a new cell. This makes cellular proliferation a metabolically demanding process, employing a large amount of biomass and energy. The commitment of a cell to proliferate is therefore strictly dependent on its metabolic status and nutrient availability . Cell cyAlready from early studies, it emerged that cells display higher rates of glucose uptake and lactate production during the logarithmic growth compared to non-proliferating phases ,121. In However, the metabolic slowdown is not a general hallmark of dormant cells. Primary human fibroblasts retain a high metabolic state when induced into quiescence, sustaining the breakdown and re-synthesis of lipids and proteins as a “self-integrity conservation” strategy and supporting ECM production. Under a dormant phenotype, these cells, thus, retain a high rate of glucose consumption, sustaining all the branches of central carbon metabolism activity, with a specific increase in the backwards fluxes from pentose phosphate pathway (PPP) to glycolysis and from α-ketoglutarate to citrate in the TCA cycle . SimilarTherefore, dormancy is an adaptive strategy exploited by different organisms and cell types to survive harsh conditions or perform definite activities, each of which is characterized by specific metabolic requirements beyond the simple regulation of cellular proliferation and cell cycle progression. This implies that, although a G0 cell cycle arrest generally characterizes cell dormancy, this phenomenon is associated with different metabolic adaptations, depending on the cell type and on the purpose of the quiescence-entry strategy.For a long time, tumor growth was considered to be mainly sustained by the so-called “Warburg metabolism” (or “aerobic glycolysis”), characterized by an increase in glucose uptake and enhanced lactate production even in the presence of adequate oxygen concentration . However® DiD) retention model and performing whole-transcriptomic profiling (mRNA-Seq) of four different breast cancer subtypes, Quayle and collaborators identified a “quiescent breast cancer cell transcriptome” associated with late recurrence that emerged to be enriched with gene products involved in OXPHOS -glucose tracing experiments demonstrated an increased OXPHOS in melanoma brain metastases compared to primary melanomas and extra-cranial metastases. Treatment with the OXPHOS inhibitor IACS-010759 reduces the risk of brain metastasis development in a mouse model [Moreover, RNA seq analysis, direct metabolite profiling, and in vivo [U-se model .Growing evidence describes the metabolic flexibility of cancer cells that undergo metastatic travel depending on the type, location, and stage of the primary tumor and the nutritional composition of the respective sites of metastasis. This great heterogeneity in metabolic adaptations makes quiescent metastatic cells particularly difficult to target and eradicate. A deeper understanding of the complexity of these processes is needed to enable a practical approach against dormant metastatic cells.The term CSCs defines the cellular subpopulations within the tumor mass possessing unique stem-like features, such as self-renewal capability, differentiation into multiple cell types, and high resistance to stress and apoptosis . CSCs plThe metabolic profile of dormant cells reflects the function they perform during tumorigenesis. In this line, data are still controversial about the differences between the metabolic states of quiescent CSCs and their differentiated progeny . Indeed,Differently, in colorectal cancer models, CD133+ CSCs exhibit increased expression of the glycolytic and TCA cycle pathways components, together with a drastic increase in nucleoside mono- and diphosphate levels. Moreover, colorectal CSCs also display a strong intracellular accumulation of free amino acids, suggesting a drastic inhibition of protein synthesis . In tripBesides relying on either glycolysis or OXPHOS, emerging evidence proves that glutamine is a crucial energy source for CSCs ,190. In Recent insights into the hallmarks of CSCs suggest that merely targeting stemness features is not sufficient to eliminate the CSCs population and prevent tumor relapse. However, targeting the metabolic adaptations supporting the quiescent status of CSCs could be a more powerful strategy than inhibiting the CSCs stemness directly to overcome drug resistance and tumor relapse.Cancer therapy often determines the emergence of drug-resistant clones, eventually leading to therapy failure. While some tumors are intrinsically insensitive to anti-cancer therapies due to pre-existing resistance factors (intrinsic resistance), others become resistant during drug treatment (acquired resistance) . Therapyhigh/ERlow therapy-resistant cells characterized by defective mitochondrial oxidative metabolism and autocrine IL-6 production. Interestingly, chronic treatment of these resistant cells raises the levels of paracrine IL-6 expression and fosters the exit from quiescence via an IL6/Stat3/Notch3-mediated induction of mitochondrial activity. IL6/Stat3 blockade by Tocilizumab restores HT sensitivity in resistant cells and suppresses the generation of CD133high cells, thus preventing the self-renewal of resistant CD133high CSCs [Some studies showed that chemotherapy-resistant cells mostly rely on OXPHOS in keeping with the selection of a quiescent subpopulation . For exaigh CSCs . The samigh CSCs .Despite these signs of progress, up to now, very little is known about the metabolic alterations underpinning the acquisition of a drug resistant-dormant phenotype. The difficulties in defining an overall therapy-induced dormancy metabolic profile are due to the heterogeneity of the metabolic patterns achieved by dormant cells to overcome the specific mechanism of action of cytotoxic agents they are exposed to.lowAutophagyhigh phenotype. In contrast, a transition to a Pfkfb3highAutophagylow phenotype is observed during metastatic relapse, suggesting that Pfkfb3, and hence, the glycolytic metabolism, play a critical role in promoting the escape of cancer cells from dormancy and reactivating proliferative programs [Alongside the aforementioned plethora of intracellular and extracellular stimuli governing cancer cell quiescence, the activation of specific metabolic programs may favor the escape from a dormant phenotype in tumor cells. Interestingly, La Belle Flynn and collaborators determined a key role for the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3) in driving the balance between dormancy and awake in breast cancer cells. Indeed, dormant cells display a Pfkfb3programs . Otherwiprograms . Other eprograms . In a brprograms .In conclusion, despite the increasing knowledge accumulated in recent decades about the metabolic pathways sustaining cancer progression, so far, very little is known about the metabolic adaptations supporting dormant cells awake. Data describing the metabolic profile of awaked tumor-initiating cells are still limited, likely due to the lack of adequate experimental models to study this phenomenon. Exploring the metabolism of these cells will be helpful to underscore pathways supporting tumor re-growth, paving the way for the development of targeted therapies for the clinical benefit.Strategies to eradicate tumor cells rely on the ability of anti-cancer therapies to affect cell proliferation. Otherwise, dormant quiescent cells may be targeted through three different approaches: (i) maintaining cell dormancy to keep cells inactive and not proliferating; (ii) “awakening” or inducing the proliferation of dormant slow-cycling resistant cells, thus allowing the use of anti-proliferative drugs; (iii) eradicating dormant cells while they are still quiescent .The first strategy is based on dormancy maintenance to elude tumor re-growth. This approach is mainly centered on activating the p38 MAPK pathway and inhibiting the ERK1/2 pathway to guarantee the preservation of anti-proliferative features by cancer cells . This meDifferently, the awakening of dormant quiescent cells is expected to sensitize the newly cycling cells to anti-proliferative therapy. However, this approach is still controversial, since the reactivation of cell proliferation could worsen the tumor aggressiveness. For instance, in an HNSCC model, a MAPK p38α/βT-dependent dormancy phenotype is induced by TGF-β stimulation. Systemic inhibition of TGF-β-RI or p38α/β activities awakens dormant cells and increases the metastatic burden in the liver, spleen, and bone marrow . HoweverA third, different approach could be eradicating cancer cells while still in the dormant state by exploiting their quiescence status. To this end, several strategies can be pursued based on the specific biology of dormant cells. Bcl-2/Bcl-XL inhibitors, such as ABT-737, effectively eliminate quiescent/slow-proliferating cells expressing high levels of anti-apoptotic proteins . As prevThe metabolic heterogeneity of dormant cells makes specific metabolic approaches feasible to eradicate quiescent tumor cells. In addition to the aforementioned VLX600, different metabolic drugs, already tested or approved as anticancer treatments, could represent additional therapeutic options. In particular, the frequently observed OXPHOS-dependent dormant cells could be targeted with mitochondrial respiratory chain inhibitors such as Metformin or Phenformin in various tumor types. In contrast, an approach based on glycolytic inhibitors for the dormant cells which rely on the Warburg metabolism could be pursued. In addition, other metabolic pathways, such as those involving glutamine, lipids, and autophagy, are potentially targetable in dormant cancer cells, mainly based on the recent success of the use of glutaminase and lipid transporter inhibitors or autophagy blockers .In this perspective, preclinical studies show the efficacy of targeting metabolic pathways to fight dormant cancer cells through the three mechanisms mentioned above . For exaThe knowledge of the metabolic features governing cancer cell dormancy entry/escape may be significant in developing effective targeted therapies. Even though, currently, it is premature to hypothesize therapeutic treatments based exclusively on a metabolic approach, the use of metabolic drugs could be of great benefit if used in combination with traditional therapies.Cancer dormancy represents an adaptive strategy that accompanies and sustains all the steps of tumor progression, allowing the survival of cancer cells in the hostile conditions they need to deal. The metabolic adaptations achieved by dormant tumor cells depends on the characteristics of the tissue of origin, environmental nutrient availability, and stromal influence, but share the common feature of slow-proliferating cells. In this context, the metabolic changes in dormant cells are often directed towards OXPHOS and ROS scavenging to ensure a sufficient energy supply under a low proliferation rate. The addiction of dormant tumor cells to specific metabolic pathways represents a helpful druggable weakness for therapeutic purposes. Future investigations may confirm that altered energy metabolism represents an attractive vulnerability to hinder different steps of tumor progression, including the maintenance of cancer dormancy or tumor relapse.


Improving speed and image quality of Magnetic Resonance Imaging (MRI) using deep learning reconstruction is an active area of research. The fastMRI dataset contains large volumes of raw MRI data, which has enabled significant advances in this field. While the impact of the fastMRI dataset is unquestioned, the dataset currently lacks clinical expert pathology annotations, critical to addressing clinically relevant reconstruction frameworks and exploring important questions regarding rendering of specific pathology using such novel approaches. This work introduces fastMRI+, which consists of 16154 subspecialist expert bounding box annotations and 13 study-level labels for 22 different pathology categories on the fastMRI knee dataset, and 7570 subspecialist expert bounding box annotations and 643 study-level labels for 30 different pathology categories for the fastMRI brain dataset. The fastMRI+ dataset is open access and aims to support further research and advancement of medical imaging in MRI reconstruction and beyond.  Among the myriad applications of machine learning in medical imaging being explored, deep learning-based MRI reconstruction is showing considerable promise and is moving towards clinical impact.Magnetic resonance imaging (MRI) is a widely utilized medical imaging modality critically important for a broad range of clinical diagnostic tasks including stroke, cancer, surgical planning, acute injuries, and more. Machine learning (ML) techniques have demonstrated opportunities to improve the MRI diagnostic workflow particularly in the image reconstruction task by saving time, reducing contrast, and leading in cases to FDA-cleared solutions5, MRNet6, OAI7, SKM-TEA8, and mridata.org are available to empower ML-related research. Also, various datasets can be found in multiple medical image research challenges, including MC-MRREC and RealNoiseMRI. Most of these datasets only provided reconstructed MRI images  or limited amount of raw data. Thus, large datasets of raw MRI measurements are generally not widely available. To address this need and facilitate cross-disciplinary research in accelerated MRI reconstruction using artificial intelligence, the fastMRI initiative was developed. fastMRI is a collaborative project between Facebook AI Research (FAIR), New York University (NYU) Grossman School of Medicine, and NYU Langone Health which includes the wide release of raw MRI data and image datasets9. While the fastMRI data has enabled exploration of ML-driven accelerated MRI reconstruction11, there is a lack of clinical pathology information to accompany the imaging data which has limited the reconstruction assessment approaches to validate quantitative metrics such as peak signal-to-noise ratio (pSNR)/structural similarity index measure (SSIM), leaving important questions regarding how various pathologies are represented in ML-based reconstruction unanswered12. For instance, low sensitivity and stability to clinically relevant features stall their clinical-aware applications14.ML-based MRI reconstruction approaches often require data from “raw” fully sampled k-space datasets in order to generate ground truth images. Public MRI datasets like Calgary-Campinas Public DatasetIn this paper, we present wide availability of a complementary dataset of annotations, fastMRI+, consisting of human subspecialist expert clinical bounding box labelled pathology annotations for knee and brain MRI scans from the fastMRI multi-coil dataset: specifically encompassing 16154 bounding box annotations and 13 study-level labels for 22 different pathology categories on knee MRIs, as well as 7570 bounding box annotations and 643 study-level labels for 30 different pathology categories on brain MRIs. This new dataset is open and accessible to all for educational and research purposes with the intent to catalyse new avenues of clinically relevant, ML-based reconstruction approaches and evaluation.9. The images used in this study were directly obtained from the fastMRI dataset, reconstructed from fully sampled, multi-coil k-space data (both knee and brain). The fastMRI dataset was managed and anonymized as part of a study approved by the NYU School of Medicine Institutional Review Board. Image reconstruction was performed by inverse Fast Fourier Transform of each individual coil and coil combination with root sum square (RSS) for the purpose of creating pre-annotation images in fastMRI+. The reconstructed images were subsequently converted to DICOM format for human expert reader (radiologist) annotation.The fastMRI dataset is an open-source dataset, which contains raw and DICOM data from MRI acquisitions of knees and brains, described in detail elsewhereAnnotation was performed using a commercial browser-based annotation platform  which allowed adjustment of brightness, contrast, and magnification of the images. Readers used personal computers to view and annotate the images using the mentioned annotation platform.A subspecialist board certified musculoskeletal radiologist with 6 years in practice experience performed annotation for the knee dataset and a subspecialist board certified neuroradiologist with 2 years in practice experience performed annotation for the brain dataset. Annotation was performed with bounding box annotation to include the relevant label for a given pathology on a slice-by-slice level. When more than one pathology was identified in a single image slice, multiple bounding boxes were used.16. Effort was made to try to include all the pathology within the bounding box while limiting the normal surrounding anatomy. If the examination contained significant clinically limiting artifacts, then the annotation for “Artifact” was added as a study-level label. In these instances, an interpolation tool was used in which the first and last slice were each labelled and the user interface interpolated the labels on intervening slices. If no relevant pathology was identified on an examination, no labels were provided.All 1172 fastMRI knee MRI raw dataset studies were reconstructed and clinically annotated for fastMRI+. Each knee examination consisted of a single series (either proton density (PD) or T2-weighted) of coronal images where bounding box labels were placed on each slice where representative pathology was identified18. As in knee examinations, effort was made to try to include all the pathology within the bounding box while limiting the normal surrounding anatomy. In some cases, the pathology or normal anatomic variant displayed within a given examination was so extensive or diffuse that a study-level label was used to characterize the relevant images or the entire exam inclusive of the finding . The study-level label, in these instances, replaced the use of a bounding box. If no relevant pathology was identified on a given examination, no labels were provided.A sub selection of 1001 out of 5847 fastMRI brain MRI raw dataset studies were selected randomly for annotation. Each brain examination included a single axial series  where bounding box labels were placed on each image in which representative pathology or normal anatomical variant was identifiedNote there are several limitations to this dataset that bear acknowledgement. First, while the annotators are subspecialist radiologists in practice at leading academic medical centers, the lack of multiple annotators/repeated annotations to determine inter-rater/intra-rater reliability metrics or ensure consensus agreement is a limitation and should be considered in the use of these labels. Further work may include multiple annotations by multiple readers to further refine the clinical labels applied in fastMRI+. Additionally, the fastMRI knee MRI raw dataset contained only coronally acquired series while the brain MRI dataset contained only axially acquired series, each in a variety of pulse sequences and coils. Most knee/brain pathologies that are visible in the non-coronal/non-axial planes are also visible in coronal/axial planes, though not as well seen or as well characterized. For instance, patellofemoral cartilage in the knee and optic neuritis in the brain. While sufficient for annotation, it is important to note that true diagnostic interpretation in MRI for the included pathologies typically demands multi-sequence and multi-planar images for clinically accurate interpretation. What is more, only binding boxes indicating knee and brain diseases were exported and reported in this work which may limit the research applications of this dataset. Full segmentation of structures would be more laborious and would be a potential subject of future work. Thus, the annotations provided by fastMRI+ may be incomplete. In the future, raw MRI datasets containing fully sampled multi-planar and multi-sequence data would enable optimal clinical annotation.Label distribution analysis was conducted for both knee and brain datasets showing detailed label descriptions at the same time. Table 9. The annotation files (knee.csv and brain.csv) can be accessed from both fastmri-plus Synapse repository19 and fastMRI-plus GitHub repository (https://github.com/microsoft/fastmri-plus) in CSV formats. Four CSV files are included in the ‘Annotations’ folder. File names of all radiologist-interpreted dataset are stored in knee_file_list.csv and brain_file_list.csv, respectively. Annotations are contained in knee.csv and brain.csv. In each annotation CSV file, the file names  are aligned with the naming in the fastMRI dataset. For each annotation, file name, slice number, bounding box information, and disease label are provided. The bounding box information includes four parameters, x, y, width (pixel), and height (pixel), representing the x and y coordinates of the upper-left corner, the width and height of the bounding box. Unit of the bounding box parameters is ‘pixel’. Study-level labels are marked as ‘Yes’ in column ‘Study Level’ for slice 0 of the corresponding subjects with no specified bounding box information.We created separate annotation files for the 1172 validation knee datasets and 1001 brain datasets, all based on the fastMRI source datahttps://docs.md.ai/). Creation and publication of fastMRI+ code repository followed standard practices with release of open-source software. Specifically, files with annotations and associated tools and scripts were managed source code control, continuous integration tests, and code/data reviews.A board-certified radiologist with 10 years of experience reviewed the overall quality of the MRI image dataset prior to annotation and clinical evaluation was performed by two additional board-certified subspecialist radiologists. We cleaned and validated raw annotation files following instructions from MD.ai Documentation (20) in order to configure a classification or object detection problem. The open-source repository also contains an example Jupyter Notebook (‘ExampleScripts/example.ipynb’) of how to read the annotations and plot images with bounding boxes in Python.The bounding box information can be used to plot overlaid bounding boxes on images, as shown in Fig.