Humans have a limited postinjury regenerative ability. Therefore, cell-derived biomaterials have long been utilized for tissue repair. Cells with multipotent differentiation potential, such as stem cells, have been administered to patients for the treatment of various diseases. Researchers expected that these cells would mediate tissue repair and regeneration through their multipotency. However, increasing evidence has suggested that in most stem cell therapies, the paracrine effect but not cell differentiation or regeneration is the major driving force of tissue repair. Additionally, ethical and safety problems have limited the application of stem cell therapies. Therefore, nonliving cell-derived techniques such as extracellular vesicle (EV) therapy and cell membrane-based therapy to fulfil the unmet demand for tissue repair are important. Nonliving cell-derived biomaterials are safer and more controllable, and their efficacy is easier to enhance through bioengineering approaches. Here, we described the development and evolution from cell therapy to EV therapy and cell membrane-based therapy for tissue repair. Furthermore, the latest advances in nonliving cell-derived therapies empowered by advanced engineering techniques are emphatically reviewed, and their potential and challenges in the future are discussed. Various diseases and accidents could lead to tissue loss and organ failure. In such situations, cells proliferate and differentiate to regenerate lost cells or tissues, along with the synthesis and resolution of extracellular matrix and subsequent modulation of fibrosis and scar tissue formation. In contrast to nonmammalian vertebrates such as salamanders, in which the capability of scar-free repair and multiple organ regeneration are maintained throughout their life, humans have a limited ability for self-regeneration, which is progressively lost throughout our life. After injury, only a few organs, such as the liver and blood, can completely regenerate in adults, while in most other tissues, fibrosis or scar formation develops as a consequence of the recession of the regenerative capability .Repairing, replacing or regenerating cells, tissues and organs is the key to regenerative medicine to restore impaired function . Cell-deIn comparison with the enduring popularity of research on EV therapy in replacement of cell therapy, the cell membrane technique is a newcomer in the field of regenerative medicine. By virtue of its excellent performance in fighting infection and tumours , 13, celAmong various cell therapies, stem cell therapy is one of the best-known and most representative methods applied to various diseases.Two major stem cell types, pluripotent and multi-/unipotent stem cells, are widely studied in the field of stem cell therapy. The former, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can differentiate into all cell types in humans, while the latter only has limited differentiation potential .Haematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and stem cells derived from foetal tissues, as multipotent stem cells, were the first to be applied in clinical treatment. Bone marrow transplantations were initially conducted in the 1950s to treat patients with haematological cancers for restoration of a fully functional circulation system through the efficacy of HSCs contained in the marrow . SimilarThe second generation of stem cell therapies was developed based on the further understanding of the capacity of pluripotent stem cells (PSCs), which are represented by human embryonic stem cells (hESCs) and iPSCs. With broad potency to differentiate into every cell type in the body, PSCs could be indefinitely expanded in vitro  for clinAccumulating evidence from the trials above has indicated the favourable translational potential of these techniques. However, their developments have been constrained by ethical and safety concerns, as well as the limited source of therapeutic cells. The use of foetal stem cells is limited not only by controversy and ethical concerns regarding the procurement of foetal tissue but also by the difficulty of isolating a sufficient number of stem cells from every foetal donor with stemness loss during large-scale culture in vitro. Such a situation has made clinical translation of therapies relying on foetal stem cells difficult . MoreoveTherefore, among these therapies, the use of MSCs in regenerative medicine has several potential advantages: fewer ethical and safety concerns and more available sources, including umbilical cord tissue, fat tissue, central nervous system tissue and bone marrow. MSCs are capable of differentiating into multiple cell types, such as osteoblasts, chondrocytes, and myocytes, to remodel bone, cartilage and vascular tissue . UnfortuGiven that the functional improvements observed in stem cell therapies might result from paracrine actions, the concept of regenerative medicine has been gradually extended from the original concept of regeneration to replace injured tissues with cell-dependent, nonspecific effects covering trophism or immunomodulation . Consist. After receiving gene therapy against X-linked severe combined immunodeficiency via transplantation of retrovirus-transduced CD34+ haematopoietic cells, four out of nine patients developed leukaemia within 31–68 months [Exogenous proteins can be introduced into cells by exogenous gene expression via virus-mediated transduction, and the cellular production of endogenous proteins eliciting reparative effects  could be enhanced through similar approaches. However, risks are associated with commonly used RNA-based retroviruses, as they might promote proto-oncogene expression or disrupt the function of tumour suppressor genes by integrating into their sequences, which leads to insertional mutagenesis considering the longevity of stem cells in vivo8 months . Self-in8 months . The uti8 months  is chall8 months . HoweverOther engineering approaches have recently been developed to enhance the efficacy of stem cell therapy. For example, the application of hydrogel-tethered interferon-gamma (IFN-γ) could increase the immunomodulatory functions of encapsulated MSCs and avoid the need for ex vivo manipulation. These MSCs could halt T-cell proliferation and dendritic cell differentiation, accelerating healing of colonic mucosal injuries  Fig. 2BB2B.However, a phase IIb trial of brain damage treated with modified MSCs transfected with a Notch intracellular domain revealed no statistically significant enhancement of endogenous NSC differentiation . In addiThe risk of pulmonary embolism and tumorigenesis needs to be defined , as evidIn addition to stem cells with cell survival promotion, immunomodulatory and angiogenic capacities, some other cells with similar or more regenerative abilities are adopted in tissue repair. For example, hepatocytes can rapidly enter the cell cycle when injuries occur in the liver and thus exert a better regenerative effect on liver failure. Because of the lack of donors, hepatocyte transplantation has been used intensively in the bridging process of orthotopic liver transplantation. However, these therapeutic cells might be transplanted to a critical environment for survival and expansion. Macrophages activated by cellular necrosis and apoptosis might induce hepatocyte replicative senescence, which impairs the therapeutic effect of hepatocytes .For the reasons mentioned above, the inflammatory response modulation of tissue repair has been increasingly studied. When injury occurs, the inflammatory response is activated by the local release of chemokines by innate immune cells recruited to the injury site, which contributes to the tissue repair response. The degree and duration of the response determine the final outcome. If the proinflammatory response does not fade away in a timely manner, the homeostasis of the injured tissue would not be restored, and tissue function would be further debilitated . The theSince the large-scale application of cell therapy still faces numerous barriers, researchers are trying to search for novel approaches to achieve tissue repair. EV therapy appeared at the right time, as it is safer, controllable by eliminating the disadvantages of living cells, and more convenient for efficacy enhancement by bioengineering techniques .As mentioned above, cell therapies mainly exert their therapeutic effects via secretion of paracrine factors. In addition to common secretory pathways, proteins and RNAs can be secreted and delivered as vesicles generated from the plasma membrane, which are called extracellular vesicles. EVs play important roles in promoting in vivo tissue repair by maintaining tissue homeostasis and modulating several physiological pathways through intercellular communication. As a repair component of the stem cell secretome, EVs have potential as a therapeutic tool in the field of regenerative medicine . TherefoBased on size, density, manner of release from cells, etc., EVs can be divided into three subgroups: exosomes, microvesicles, and apoptotic bodies . AlthougStructurally, EVs exert intercellular communication through their membrane and cargos in the lumen. The EV membrane maintains features from its original cell membrane and thus could mediate the interactions between EVs and target cells . With thStudies on the tissue repair potential of EVs can be traced back to 2009, when EVs secreted by MSCs were expected to exert major therapeutic effects in a kidney injury model . TherefoIn addition to EVs derived from stem cells, EVs from other cells are employed in the repair process , inheritXenogenic cell-derived EVs are also surprisingly able to induce tissue repair. EVs from newt limb explant cells (A1 cells) were similar to mammalian EVs in diameter, structure, and proteins from the lumen and membrane surface. Nevertheless, both RNAs and proteins contained in A1-EVs showed significantly higher levels per vesicle. Numerous unique RNAs and proteins were found in these EVs, many of which were elements expressing nuclear receptors, membrane ligands and transcription factors. After the administration of these A1-EVs to mammalian cardiomyocytes, increased gene expression of survival promoting the PI3K/AKT pathway was detected, which prevented the oxidative stress-induced apoptosis of cardiomyocytes .Compared to cell therapy, EVs have abundant advantages as they are nonliving entities. EVs could provide similar therapeutic effects as cell therapy and also overcome their limitation of poor survival after implantation, as they are directly internalized by the target cells . In addi6 cells is 1 to 4 μg/day, which could barely meet the demand for a dosage of 100 to 500 μg per patient of EVs in a clinical trial [However, there are still aspects of EV therapy that have yet to be improved. EVs are quickly cleared in the liver, lungs, and spleen with a short half-life in minutes after systemic administration. Rapid clearance of EVs from the injection site also indicates that very limited improvement could be achieved through local administration , which lal trial .Only a small amount of EVs can reach the target site and exert therapeutic efficacy, which impairs the effects of EV therapy and raises the required yield of EVs . TherefoTargeting EVs to specific tissues and organs could be improved by loading targeting molecules in the lumen or on the membrane of EVs. The molecules modified on the membrane could interact with specific receptors on target cells or components in the microenvironment surrounding the target . For exaApart from physical  or chemiIn addition to membrane modification, exogenous guidance could lead to targeted delivery of EVs to action sites. For instance, Kim et al. fabricated iron oxide nanoparticle (IONP)-incorporated EVs from IONP-treated MSCs to enhance the targeting ability of EVs, in which IONPs acted as a magnet-guided navigation tool, significantly increasing the EV accumulation in the injured spinal cord under exogenous magnetic guidance  Fig. 3BB3B.The yields of EVs and therapeutic cargos in EVs could be enhanced by modulation of EV-secreting cells by culturing them in stress-inducing conditions –81 or tr7 macrophages, while the same number of macrophages in the control group only produced 0.06 mg of EVs [Referring to the former approach, Li et al. applied cytochalasin B in macrophages to relax the interaction between the cytoskeleton and membrane and thus to stimulate macrophage-derived EV secretion. This approach was relatively convenient and had a high efficiency: approximately 0.3 mg of EVs could be isolated from 10g of EVs . Yang etg of EVs .Regarding the transfection method, Ran et al. demonstrated that via transduction of myostatin propeptide with its inhibitory domain fused into the second extracellular loop of CD63, the propeptide could be overexpressed and anchored to the EV membrane, which significantly enhanced the efficacy of EVs on myostatin inhibition, accelerating muscle regeneration and growth . In contEither through culturing cells under IFN-γ stimulation or through genetic cell engineering, Su et al. obtained a high concentration of PD-L1 in EVs, which suppressed T cell activation after specific binding to PD-1 on the cell surface, significantly speeding up wound contraction and re-epithelialization during the inflammation phase .Another novel technique has been developed to enhance EV cargo in recent years , 77, 84.The present understanding of the mechanism of intracellular EV cargo release is limited. As a large proportion of EVs degrade in the endolysosomal system after internalization, recent studies have aimed to modulate the cell endocytic capacity and surface modifications of EVs to optimize intracellular trafficking and enhance endolysosomal escape . StrategRecently, it was reported that in an acidification-dependent manner, a proportion of internalized EVs could integrate with the limiting membrane of endosomes and lysosomes, leading to the exposure of their cargos to the cytoplasm, which might inspire new approaches .EV uptake by target cells could be enhanced by locally administering EVs at the site of injury, while slowing the clearance of EVs from the administration site is also critical. Thus, several strategies have been established to achieve sustained release of EVs at the injured site to enhance EV presentation to recipient cells by utilizing multiple biomaterials, including hydrogels incorporating hyaluronic acid , alginatRecent progress has been made in the spatial and temporal release of EVs. Silva et al. applied allogenic EVs from ASCs with a thermoresponsive gel to a porcine fistula model. After gelling at body temperature, the gel retained EVs in the entire fistula tract, completing 100% fistula healing . NotablyEV therapies have various advantages in tissue repair and regenerative medicine endowed by the functions of the EV membrane and cargos in its lumen, which mimic the capacities of their original cells to exert intercellular communication. However, there is still a long way to go for the clinical translation of EV therapies, considering the complexity of EV isolation and the uncertainty of various nontherapeutic factors in EVs. On the one hand, there is no consensus on the optimal approach for EV isolation, as each isolation technique has its own advantages and limitations . On the Cell membrane coating technology was first reported in 2011. By transferring the outermost layer of a cell directly onto the surface of an NP, Zhang’s group established this technique, which utilized entire cell membranes as the NP coating material. This technique could faithfully preserve the complexity of the membrane, including all of its lipids, proteins, and carbohydrates, which enable the membrane‐coated NP to exhibit as many properties as the source cell. With this technique, red blood cells (RBCs) became the first source of membrane material . RBCs arSince then, membranes from multiple types of cells, including RBCs, platelets (PLTs) , leukocyIn the past, cell membrane coating technology was mainly utilized in the fields of infection and tumour treatment. Recently, cell membrane-based therapy has extended its application to tissue repair with a variety of cell membranes and bioengineering technologies employed.The use of cell membrane technology in tissue repair was previously explored to solve the problems in stem cell and stem cell-derived EV therapy. As an alternative approach to EVs, packaged secreted factors from MSCs were coated with stem cell membranes , 99. TheSimilarly, in addition to stem cells, RBC-coated NPs that carry beneficial regenerative factors from MSCs can be applied to repair tissue. In one situation, RBC membrane-coated NPs achieved increased blood stability with lower internalization by macrophages and were able to remain in the liver, where the NPs could promote liver cell proliferation and ultimately mitigate carbon tetrachloride-induced liver failure in a mouse model .Realizing that paracrine factors are the key therapeutic element in stem cell-mimetic therapy, Zhang et al. introduced a size-variable artificial stem cell spheroid (ASSP) technique, combining the paracrine functions of three-dimensional (3D) SSPs for vasculature regeneration. First, for generation of a hypoxic microenvironment for enhanced proangiogenic factor secretion, MSCs were induced to aggregate into 3D spheroids. Then, depending on the disease and corresponding action site, SSP-secreted factors were integrated into different types of membranes selected to exert their function, forming MPs/NPs with MSC/RBC-PLT hybrid membrane–derived surface coatings. By virtue of the easily controllable size of the ASSP particles, the local administration of ASSP MPs and systemic delivery of ASSP NPs could provide superior revascularization effects in the ischaemic tissues of hindlimb ischaemia models and MI models  Fig. 4AA4A.Fig. 3O4 NPs into rats with articular cartilage defects, where the NPs encapsuling the cartilage regeneration-promoting drug kartogenin were able to more efficiently promote the regeneration of a layer of hyaline-like cartilage with an organized structure [Considering the possible risk of the direct use of native cell secretions containing various unknown factors, some verified effective elements from paracrine factors (such as certain miRNAs) and drugs were loaded into cell membranes to mediate the therapeutic effects. Yao et al. reported the self-assembly of MSC membrane-camouflaged miRNA-loaded mesoporous silica NPs. The MSC membrane helped NPs target ischaemic cardiomyocytes, where miRNA inhibited the translation of apoptosis-associated proteins. As a result, these NPs promoted the proliferation of cardiomyocytes and thus led to preservation of surviving myocardium and cardiac functions in the MI mouse model . Zhang etructure .The abovementioned evidence indicates that the therapeutic effect in tissue repair could be achieved without stem cell membranes or paracrine signalling. Therefore, more combinations of various types of membranes from RBCs, PLTs or leukocytes and therapeutic cargos have been assessed.In atherosclerosis models, either rapamycin‐loaded NPs with RBC or PLT membranes reduced macrophage‐mediated phagocytosis and enhanced plaque accumulation of NPs, which ultimately attenuated disease progression , 110. LuLeucocyte membranes were subsequently used in cell membrane-based therapy. In addition to their inflammation targeting ability, leukocytes have shown the capacity to bind and neutralize proinflammatory cytokines, which could trigger immune activation and tissue injury. Zhang et al. integrated neutrophil membranes onto polymeric cores with the antigenic exterior and associated membrane functions of inherited neutrophils, which made them ideal decoys for biological molecules targeting neutrophils. The coating membrane could neutralize proinflammatory cytokines and thus suppress synovial inflammation and provide powerful chondroprotection against joint injuries . By loadRecently, more types of cell membranes have been applied in NP coatings in addition to those of stem cells and haematocytes. To overcome the low drug accumulation in the substantia nigra and striatum, which is the main challenge of Parkinson disease (PD) treatment, Liu et al. camouflaged NPs with membranes from MES23.5, which is a hybrid cell line derived after fusion of rat embryonic mesencephalon cells with mouse neuroblastoma. Possessing the biofunctional surface of substantia nigra dopaminergic neurons, these membrane-coated NPs could effectively target microglia in the substantia nigra through interactions between their surface vascular cell adhering molecule 1 (VCAM1) and α4β1 integrin on microglia and provide NPs with the ability to modulate microglial overactivation for PD treatment . A blood2O2 in inflammatory tissues. By macrophage membrane coating, the ROS-responsive property of NPs enabled specific payload atorvastatin release after being led to inflammatory tissues, attenuating the proinflammatory efficacy of macrophages and the formation of foam cells in atherosclerosis [2O3 magnetic NPs [Additionally, the targeting capacity of native cell membrane-coated NPs could be further enhanced by combining nanotechnology in cargo design. The inflammatory response in multiple tissue injuries could overproduce reactive oxygen species (ROS) –123. Thrclerosis . A similclerosis . Promoteetic NPs  , interleukin‐6 (IL‐6), and tumour necrosis factor alpha (TNF‐α) receptors, which are induced by plasmid transfection. Combined with MI-protecting miR199a‐3p, these NPs could suppress inflammation, prevent hypoxia‐induced apoptosis, promote cell proliferation and inhibit cardiac fibrosis in mice with MI . Similar2 into the PLT membrane for NP coating could take advantage of the native MI‐homing capacity of the PLT membrane and the prostaglandin E2 receptors overexpressed in the microenvironment of cardiac I/R injury, allowing NPs to target delivery of therapeutic cargos to the injured heart [Strategies for chemical modification are mainly based on lipid‐lipid interactions with the membrane and various conjugations to active sites on the membrane, such as primary amine and thiol residues of proteins and hydroxyl residues of polysaccharides on the cell membrane . Specified heart . The RBCed heart .Furthermore, multistage targeting capacity could be achieved via various modifications. Han et al. developed membrane-coated solid lipid NPs with attachment of a triphenylphosphine (TPP) cation and a 29 amino acid peptide derived from RVG (RVG29) to the surface of the membrane. The decoration of RVG29 on the membrane surface endows the NPs with the ability to cross the BBB for specific targeting to neurons. TPP further led the NPs to mitochondria after entering neurons, where loaded elements had the most effective therapeutic effects on AD symptoms , 139.Chemical engineering could also be used in therapeutic element loading. Wu et al. decorated macrophage membrane-coated NPs with an apolipoprotein A-I mimetic peptide to inhibit oxidized low-density lipoprotein uptake and promote cholesterol efflux in intimal foam cells, which exhibited excellent antiatherosclerotic effects . Moreove2O2-cleavable phenylboronic ester linkage, RBC membrane-coated tirofiban exhibited site-specific antithrombotic effects after efficient accumulation at the injured carotid artery in a thrombosis mouse model [Under certain situations, therapeutic cargos might undermine the targeting function of the optimal coating membrane type. Chemical insertion of targeting molecules could help to mediate the corresponding function. For example, a PLT membrane was the best surface coating tool for thrombus targeting, contributed by the thrombus-homing property of PLTs. However, in the study of Zhao et al., tirofiban, as an antagonist of the PLT glycoprotein IIb/IIIa receptor, was selected for antithrombotic therapy, which might compromise the targeting capability of the PLT membrane. Therefore, the RBC membrane was functionalized with the fibrin-targeting peptide CREKA. As controlled release of tirofiban was achieved with conjugation to the nanocarrier core through an Hse model .Apoptotic cell efferocytosis could induce a phenotypic transition of macrophages towards proreparative M2 macrophages, facilitating the resolution of inflammation. NPs coated on apoptosis-featured phosphatidylserine (PS)-supplemented cell membranes could mimic crucial properties of the apoptotic cell surface, exerting anti-inflammatory effects by being engulfed by macrophages such as apoptotic cells, which could help mitigate macrophage-mediated inflammation . FurtherAlthough coated with the cell membrane, NPs might still induce an immune response in vivo after membrane camouflage is washed out. Therefore, researchers are also exploring methods to load therapeutic elements without NPs. Dong et al. directly loaded resolvin D2 (RvD2) into neutrophil membrane-derived nanovesicles, enhancing the resolution of inflammation, as RvD2 was able to incorporate into the lipid bilayer of nanovesicles by its lipid structure . In anotCell membrane-based therapy as a top-down approach has limitations due to the difficulties in controlling physical parameters such as size and homogeneity; controlling the packaging and reserve of chemically different molecules such as hydrophilic, lipophilic and amphiphilic molecules; and formulating a standardized protocol for manufacturing and preservation . MolinarAs this bottom-up approach provides more tunability in the formulation design of NPs, the physicochemical properties and biomimetic behaviour of these NPs might change under variable biological conditions. For example, based on the assumption that the protein content on the NPs was related to their biomimetic targeting capacity, Zinger et al. constructed anti-inflammatory NPs with intact structural properties and improved inflamed endothelium-targeting capability by optimizing the protein-lipid ratio at a maximum of 1:20 (w/w) in the synthetic process .Cell membrane-based therapies do not always contradict cell therapies or EV therapies, and sometimes their combination could lead to unexpectedly excellent outcomes.By combining cell membranes with cell therapies, Tang et al. showed that cardiac stem cells (CSCs) with PLT membranes fused onto their surface exhibited PLT surface markers associated with adhesion of PLTs to injury sites. The modified CSCs could thus preferentially bind collagen-coated and endothelium-denuded aortas in rat and porcine models of acute MI, enhancing their retention in the heart, where they could repair tissue and reduce infarct area. Through the conjugation of PLT membranes, this manipulation method for stem cells is convenient and safe, requiring no genetic editing of the cells, and might be applied to various other cell types . Li et aIn terms of the incorporation of cell membrane-based therapies with EVs, Zhang et al. aimed at the obstacles of poor homing efficiency and low yields of stem cell-derived EVs and enhanced the targeted delivery capacity to ischaemia-injured myocardium of MSC-derived EVs with monocyte membranes through membrane fusion. By mimicking the recruitment feature of monocytes through exclusive adhesive molecules, particularly the interaction between intercellular adhesion molecule 1 and lymphocyte function-associated Ag-1 or macrophage-1 antigen, these EVs exhibited improved efficacy for endothelial maturation promotion and macrophage subpopulation modulation and improved therapeutic outcomes in cardiac function and pathohistological changes  Fig. 5DD5D. WithAlthough nonliving cell-derived therapy, including EV therapy and cell membrane-based therapy, holds promise in tissue repair and regenerative medicine, this field is still in its early stages with multiple technical obstacles hindering its clinical translation.Although multiple novel methods and materials have been used to establish advanced EVs and cell membrane-coated NPs, there are still doubts about their ability to improve the efficacy of nonliving cell-derived therapies. Most doubts are related to two key questions: I. EVs or cell membrane? II. Native or engineered?For the selection of EVs or cell membranes before engineering, it is vital to clarify the main difference between them and their cargos. In contrast to the fact that cell membrane-based therapies are usually based on functional NPs such as cargos, EV therapies generally rely on endogenous cargos within EVs, with or without engineering. However, these EV cargos could lose therapeutic efficacy in the process of engineering; for example, EVs or exogenous cargos such as siRNA might aggregate during electroporation . AdditioWhile the function of the EV membrane could be replaced by the plasma membrane in most situations (pristine EV membrane-coated NPs are more common in tumour homologous targeting), the main advantage of EV therapies is the organized function of complex EV cargos, which is irreplaceable by the combination of known factors or drugs. Once the question of how EVs actually exert their ability in a specific injury, namely, what specific EV functions are mediated by what specific cargos, is answered, the efficacy of endogenous EV cargos could be replaced , 157. AtCombining the answers to the first and second questions, a rule for the decision to choose native or engineered EVs or cell membranes could be acquired. Depending on different tissue injuries, from some that have been studied thoroughly to those whose mechanisms are unknown, diverse therapies could be applied. The better we know an injury, the more specific therapeutic elements and more engineered methods should be utilized to repair this kind of injury to enhance the known therapeutic mechanisms. For injuries that have not been thoroughly investigated, a more natural approach might be applicable before defined therapeutic or targeted elements are found to use for the repair of these injuries.To date, no consensus on the optimal approach for EV isolation has been reached, as each isolation technique has its own advantages and limitations. Among various techniques, EV aggregation and changes in EV structure might be induced by differential ultracentrifugation, and there is a risk for coisolation of protein aggregates with other soluble factors. In size-exclusion chromatography, potential EV loss and increased processing times might be caused by the need for concentration of EV-containing medium before injection into purification columns . Similar10–1011 EVs were an effective therapeutic dosage to treat one patient with graft-versus-host disease [14 vesicles per dose might be required for the clinical application of EVs [For clinical translation, the EV dosage regimen in EV therapy has been highly contradictory, as a broad range of EV doses of 30–1300 μg/kg and 4–4000 μg/kg have been applied to vascular disease in rats and mice, respectively [ disease , while on of EVs . As a moAs a result, large-scale and standardized experiments are needed to compare these various approaches for the corresponding cell-derived biomaterials and to formulate the general dosage regimen. However, malignant cells might help in the large-scale manufacture of nonliving cell-derived therapy. Research has shown that a malignant T cell line (EL4 cells) rather than primary T cells could be used in the cell membrane coating technique, with a low risk of tumorigenesis in vivo due to elimination of tumorigenic genetic materials, which is cost‐effective and less time‐consuming than adoptive T cell transfer therapy . The utiThe development from cell therapy to EV therapy and cell membrane-based therapy for tissue repair is at the same time the evolution from a natural approach to an engineered approach with the mechanism of cell therapy efficacy fulfilled by paracrine factors being clear. In this review, we systemically summarize the utilization of multiple native or engineered EVs and cell membranes combining advanced bioengineering techniques in the treatment of various tissue injuries. Although there are still challenges for functional realization and clinical translation, the accumulation of evidence suggests that these nonliving cell-derived therapies, especially cell membrane-based therapies, have great potential in the field of tissue repair due to their strong ability to combine with novel advanced materials and methods. However, as a newcomer in the regenerative field, this kind of technique has a much greater potential than those applications mentioned above. There are multiple new fields, advanced coating techniques and biomimetic working mechanisms of cell membrane-based therapy in tissue repair and regeneration. By combining cell membrane-coating technology and advanced materials, researchers can address a variety of problems in NP applications. For example, the use of NPs usually results in endothelial cell leakiness, which is caused by interactions with the vasculature, including physical interactions between NPs and endothelial cell adherens junction proteins , 163. LeIn regard to the origin of the coating membrane, Gong et al. recently performed pioneering work to introduce intracellular membranes of organelles to NP camouflage. In their research, the outer mitochondrial membrane (OMM) from mouse livers was coated onto NPs as a representative. By protecting cells from cell death and apoptosis induced by the B-cell lymphoma protein 2 inhibitor ABT-263, which targets the OMM, OMM-coated NPs could ameliorate thrombocytopenia, which is a major adverse effect in the clinical use of ABT-263 . In tissIn-depth understanding of the mechanism for tissue repair sometimes is far more matter than innovation in engineering. Defining new targets and cells for biomimetic engineering could be meaningful via research on the microenvironment of the injured area, where various cells continuously initiate communications, and different cells might exert multiple functions with the injury response developing both in time and space. For instance, with in-depth research on the glucose-responsive insulin secretion function of beta cells, Chen et al. constructed artificial beta cells equipped with a glucose metabolism and membrane fusion system possessing a multicompartmental 'vesicle-in-vesicle' superstructure, which had the function of beta cells and showed promise for improving clinical outcomes in people with diabetes . Such a


Somatic mutations arise postzygotically, producing genetic differences between cells in an organism. Well established as a driver of cancer, somatic mutations also exist in nonneoplastic cells, including in the brain. Technological advances in nucleic acid sequencing have enabled recent break-throughs that illuminate the roles of somatic mutations in aging and degenerative diseases of the brain. Somatic mutations accumulate during aging in human neurons, a process termed genosenium. A number of recent studies have examined somatic mutations in Alzheimer’s disease (AD), primarily from the perspective of genes causing familial AD. We have also gained new information on genome-wide mutations, providing insights into the cellular events driving somatic mutation and cellular dysfunction. This review highlights recent concepts, methods, and findings in the progress to understand the role of brain somatic mutation in aging and AD. Somatic mutations are acquired alterations in the genome of an organism. Unlike germline mutations, which are typically inherited from a parent and are present throughout the body, somatic mutations arise in an organism after the single-cell zygote state and exist only in a subset of the organism’s cells. Distinct classes of somatic mutation include single-nucleotide variants (SNVs); aneuploidy; copy number variants, including insertions and deletions; structural variants; and transposable element (TE) insertions. Somatic mutations have been studied for decades in the context of neoplasms, where they form the foundational theory of carcinogenesis , 124. MoIn contrast to germline mutations, somatic mutations are present only in a subset of cells, forming a mosaic where the organism has a heterogenous composition of cellular genomes. Somatic mutations may occur in multiple cells  or may be unique to a single cell (private), depending on the mechanism and timing of their occurrence .When arising in proliferating cells, somatic mutations take a clonal pattern, with all daughter cells exhibiting the same mutation. In the context of development, a new somatic mutation would be passed on to all daughter cells, delineating a clone of that lineage. Depending on the developmental timing of the mutation, a new somatic variant may be widely distributed in multiple tissues, or may be restricted to a single tissue or even a narrower localization. In the context of neoplasia, a somatically mutated cell can have a selective growth advantage and proliferate to create a clonal population. Both developmental and neoplastic clonal somatic mutations are thus present in multiple cells and can generally be detected using bulk DNA sequencing methods, whether using whole-genome or targeted methods .When a cell specializes and is no longer undergoing cell division, it is said to be postmitotic. Surprisingly, at least some of these noncycling cells also appear to acquire somatic mutations, which are exclusive or private to that individual cell. Since these variants are not clonally expanded, they are not detectable using traditional DNA sequencing methods and require specialized methods for single-cell detection. These single-cell methods may also be applied to proliferating cell populations to define the genomic properties of individual cells. Somatic mutations may also be detected by variant-specific methods such as droplet digital PCR , 133. ReBraf V600E variant in mouse neuronal progenitors contributes to epileptogenicity  , 73, 132Cancer genome analyses have identified a number of mutational signatures . NotablyAPP) produce FAD (PSEN1) (PSEN2)  have revealed several genes showing autosomal dominant genetic inheritance. In turn, these genes have illuminated biochemical mechanisms of protein processing and folding that constitute the basis of the major paradigm for AD initiation. Germline mutations in the amyloid-β (Aβ) precursor protein gene  , 112 and (PSEN2) , 106 als (PSEN2) , 110, anAPOE ε4 allele confers the greatest risk, 80% lifetime for homozygotes  increases the lifetime risk of AD, largely through APP overexpression . However, Kim et al.   . While tTEs are mobile DNA sequences that can change location within the genome and include classic transposons  that operate via a cut-and-paste mechanism, as well as retrotransposons  that utilize an RNA intermediate and reverse transcriptase in a copy-and-paste mechanism . In humaDrosophila models of tauopathy have revealed that tau pathology is associated with increased chromatin relaxation (Studies in laxation . A pair laxation , 116. Holaxation . Direct BRAF V600E and other MAPK pathway variants. In such individuals, lesions occur in the cerebellum and basal ganglia, with corresponding clinical neurological symptoms. To further investigate the possible role of somatic mutations and histiocytosis in neurodegeneration, Mass et al. (Braf V600E in specific yolk sac erythro-myeloid progenitors that populate the brain in early development and generate microglia, the brain tissue-resident macrophages. These mice showed clonal expansion of tissue-resident macrophages and severe late-onset neurodegenerative disease, bolstering the link between somatic mutation–driven proliferation and neurodegeneration. Indeed, a diverse group of somatic variants can cause histiocytosis diseases (BRAF V600E mutations in AD brain (DNMT3A or TET2, which are cancer-associated genes that are also mutated in clonal hematopoiesis (While much of the attention on somatic mutations in AD and other neurodegenerative diseases has focused on a somatic version of familial disease genetics, clues from other disorders suggest that multiple somatic mechanisms may produce neurodegeneration. For example, certain neurodegenerative phenotypes can occur in patients with the somatic mutation–driven neoplasm Langerhans cell histiocytosis , which rs et al.  developediseases , providiAD brain , 93, smaopoiesis , 54, or Beyond the effect of variants in a single gene, the full aggregate of somatic mutations in the genome carries the potential to significantly impact cellular function and health. While sequencing technology has developed dramatically in recent years , the majSingle-cell methods are able to detect mutations that are present only in individual cells , 134, whMutational signature analysis of scWGS data from NER-deficient neurons showed an abundance of signature C above the levels seen in control neurons . SignatuThe observation of signature C mutations in human neurons that are genetically deficient in NER  indicateThe DNA damage theory of aging postulates that DNA damage contributes to genomic instability and the overall process of aging . SomaticSomatic mosaicism in the brain can range from many clonal cells to a private mutation in an individual cell. Specific clonal somatic SNVs cause neurodevelopmental diseases and may play a role in AD and other neurodegenerative diseases, though currently published studies have not established a clear role for somatic FAD gene mutations in causing AD. However, genome-wide, SNVs are increased in aging and in nucleotide repair deficiency–related neurodegeneration, where mutational signature analysis has enabled the identification of specific cellular processes involved in the generation of somatic mutations, which is of potential importance to broader cellular function in these conditions and neurodegeneration broadly. Furthermore, somatic mutations across the genome may lead to cellular harm through multiple mechanisms. Technological advances have enabled rapid progress in understanding somatic mutations in the brain, and future advances hold great promise to enable the detection of mutations at lower abundance, with higher cellular throughput for more complete analysis, and even accompanied by other cellular information , such as


Personalized treatment of cancer patients demands specific and validated biomarkers for tumor diagnosis and therapy. The development and validation of such require translational preclinical models that recapitulate human diseases as accurately as possible. Moreover, there is a need for convergence of different (pre)clinical disciplines that openly share their knowledge and methodologies. This review sheds light on the differential perception of biomarkers and gives an overview of currently used models in tracer development and approaches for biomarker discovery. Cancer is one of the leading public health care issues, which imposes a high clinical burden and requires a vast amount of economic resources . AccordiBiomarkers are characteristic biological features that can be objectively measured and indicate a normal biological or pathological process in the body. In literature, a variety of biomarkers derived from molecular, histologic, physiologic or radiographic disease characteristics can be found: blood glucose levels, protein expression, and blood pressure or tumor size, respectively. Even complex organ functions or changes in biological structures are used as medical biomarkers .Furthermore, one distinguishes between diagnostic, prognostic, predictive and therapeutic biomarkers ,8. LiterThe following examples are given to underline these different aspects of biomarkers: Patients with PD-L1 overexpression tend to have a higher response to anti-PD-L1 directed therapy. This is the case especially for non-small cell lung cancer and melanoma. However, the high responses of some patients with low expression levels complicate the use of PD-L1 as an exclusionary predictive biomarker . The valIn the field of molecular imaging, the term “imaging biomarker” is frequently but often misleadingly used. An imaging biomarker is a measurable biological variable indicative of normal or pathological conditions that is measured using imaging techniques. In accordance with this definition, radiolabeled tracers are not biomarkers themselves but rather tools. Dedicated imaging readouts  or (semi-) quantitative parameters such as standardized uptake value or binding potential serve as imaging biomarkers. Due to the non-invasiveness of imaging, imaging biomarkers can be used in sensitive (early) and specific diagnosis of diseases, monitoring of disease progression and therapy response .Molecular imaging is based on a molecular interaction between an imaging probe  and a receptor or a characteristic biological feature. In nuclear medicine, this interaction can be detected (and quantified) via the physical decay of the integrated radionuclide by suitable camera technologies, allowing for non-invasive imaging of the whole organism ,17. The Based on the radionuclide, one distinguishes between single-photon emission computed tomography  and positron emission tomography . Both methods can very sensitively visualize the interactions between targets and ligands and are able to detect picomolar concentrations of radiotracers . Due to Multimodal imaging referring to the combination of nuclear medical imaging techniques such as PET and SPECT with magnetic resonance imaging (MRI) or computed tomography (CT), adds the necessary anatomical information for correct organ (or tumor) delineation and image interpretation . CT is bEspecially in the diagnosis and treatment of cancer, it is essential to evaluate the extent and localization of the primary tumor and potential metastases, tumor heterogeneity or early treatment response. The different imaging modalities are used in clinical practice to guide surgery, biopsy or simply to detect organs affected by the disease. However, the combination of molecular imaging with (image-guided) biopsies, although being invasive, still remains necessary and is extremely valuable (c.f. 2.2).endogenous ligands or derivatives, that map metabolic (dys)functions ordrugs or (modified) model substances targeting specific disease-related proteins  orantibodies or antibody constructs directed against specific disease-associated antigens .Radiopharmaceuticals are the heart of nuclear medicine providing the interaction with and quantification of disease-related targets of interest. Mainly, radiopharmaceuticals are radioactively labeled:99mTc]Tc-MIBI, initially designed for cardiac imaging, mimics potassium (K+) for transport into cells within well perfused areas with subsequent electrostatic interaction on the mitochondria. However, besides cardiac imaging, applications range from parathyroid or thyroid imaging to breast cancer imaging. Another example is [99mTc]Tc-HEDP, which accumulates in hyperactive bone tissue by interactions of its phosphate groups with hydroxyapatite (“bone-seeking”) and visualizes pathologies with a variety of causes. Accordingly, these tracers visualize a molecular change that is not necessarily causal to the disease [111In]In-DTPA-Octreotide or [111In]In-Zevalin) PIB) PIB) . Due to 11C]PIB) ,34.68Ga]Ga-DOTA-TATE), as chelator-related alterations of biological properties are more tolerated .,46.45,46As an alternative strategy to patient samples, the availability of data from different public databases such as The International Cancer Genome Consortium (ICGC) , The CanThe success story of molecular imaging highly depends on the availability of powerful imaging probes with favorable biodistribution and imaging characteristics. Radiopharmaceuticals must reach the target of interest in a reasonable time period and with a high degree of reproducible metabolic stability. These characteristics of new imaging biomarkers need thorough validation and translation in a systematic way during tracer development to serve as decision-making tools in clinical practice .Radioligand binding assays are commonly employed during radiopharmaceutical development to determine the affinity and selectivity of a ligand for its respective target, binding kinetics and number of available binding sites. By performing binding assays for several time points, information about association and dissociation rates of the ligand from the receptor is obtained over time and optimal assay conditions for further (saturation) experiments can be acquired ,59. Semi-automated devices became recently available allowing for the assessment of binding kinetics from a single radioligand concentration in real-time: a detector, mounted on top of an inclined support, measures cell-associated radioactivity on a rotating petri-dish  in comparison with a reference region . TherebyReceptor saturation and competitive binding experiments are performed at binding equilibrium to determine affinity constants, such as Kd (equilibrium dissociation constant) or Ki (inhibitory constant). The maximal number of available binding sites (Bmax) is determined using receptor-saturating concentrations of radioligand. In addition, blocking or displacement conditions are employed to determine a specific signal by co-incubation with an excess of unlabeled competitor targeting the receptor of interest. These assays can be performed either using whole cells or membrane preparations ,63. Autoradiography, being amongst the most sensitive imaging techniques, can be performed either in vitro (incubation of tissue sections with radioligand) or ex vivo . The latter involves physiological tracer distribution and in vivo binding, whereby the radioactivity detection is carried out post mortem using dedicated phosphor storage screens or autoradiographic emulsions. Co-injection of (ex vivo) or co-incubation with (in vitro) an excess of unlabeled competitor or inhibitor targeting the receptor of interest reduces the specific signal of the radioligand. Thus, blocking or displacement experiments are used to determine specific binding . Deeper insights into pathophysiological pathways of underlying diseases can be obtained by comparison of autoradiograms with immunohistochemical staining of ideally identical or adjacent tissue sections. While autoradiograms reflect radioligand binding to tissues, immunohistochemistry proves the respective target expression. Both however provide only a snapshot, whereas in vivo molecular imaging provides real-time distribution in the living organism, showing how complementing the two different fields are. Since the interaction between tracer and target are comparable to autoradiography or IHC, molecular imaging based on tracers could also be described as in vivo pathology.The above-mentioned assays to evaluate suitable molecular tracers are first and foremost conducted using the already in the 1900s introduced, two-dimensional (2D) cell culture systems . HoweverThese drawbacks are expected to be remedied by three-dimensional (3D) cell culture, pioneered by Holtfreter, which has the potential to revolutionize drug testing and disease modeling ,68. In cIn general, multicellular tumor spheroids are characterized by a surface layer, consisting of proliferating cells, and an intermediate layer, which is connected to a perinecrotic region. This perinecrotic region contains non-proliferating hypoxic cells, which form a necrotic core—a phenomenon commonly observed when the diameter of the spheroid reaches 500 µm ,71,72. This structure not only leads to cellular heterogeneity within the spheroid and thus a more realistic mimic of in vivo tumors, but also develops a molecular gradient and penetrating barrier for drugs and metabolites, allowing researchers to conduct drug treatment tests without the use of animals. Nevertheless, 3D culturing methods still face limitations. Amongst them is the difficulty of imaging , and the fact that many assays turn out to be endpoint assays due to the need for generation of single-cell suspensions  ,74. HoweSome limitations were recently leveraged by the development of organoids, innovative 3D primary cultures that mimic the native organ microstructures and are derived from self-organizing mammalian pluripotent or adult stem cells. Histological and molecular characterization of organoid cultures has shown that organoids recapitulate the structure and function of organs and tissues . FurtherPDOs provide a model of human cancer, still representing many characteristics of the original tumor. Organoids can be used to simulate the clinical situation more appropriately, for understanding both, cancer-associated and tracer-related processes. This leads to a more pronounced comprehension of the mechanisms and potential interpretation of the clinical setting. Despite the great progress in the development of advanced in vitro models, animal models remain indispensable in cancer research. They play a critical role in the transition from fundamental diagnostic and therapeutic discoveries to human clinical trials. Murine models, in particular, contributed significantly to the understanding of the genetic basis of cancer and helped to elucidate the role of specific genes and respective mutations in cancer development and progression . Genetically modified rodents and immunocompromised mice engrafted with (human) tumor cell lines allow for studying a variety of different tumor types and imaging of those. However, these models are often criticized for their low translational value and negligible clinical significance, as they reflect human tumor biology only in some aspects . OrganoiIn general, small animal cancer models are more frequently used as (the scarcely used) large animal models. Due to their small size, short lifespan and high fecundity , these models allow for timely studies, large cohorts, ease of housing and cost-effective maintenance. In this context, the use of non-mammalian vertebrate models in cancer research such as zebrafish has become increasingly popular. Until the onset of the adaptive immune system at around seven days post-fertilization, zebrafish embryos can engraft transplanted cancer cells (zebrafish xenograft models). Moreover, genetic models of cancer in zebrafish have been developed. Aspects of human disease can be recapitulated and followed in vivo at the molecular level because cancer-related genes and signaling pathways are largely evolutionarily conserved between zebrafish and humans ,81. HoweThe chorioallantoic membrane (CAM) assay represents another embryonal, non-mammalian, vertebral model and serves as a potential alternative to rodent models. The highly vascularized chicken CAM allows for tumor cell inoculation, transplantation of tissue specimens, and vascularization of those. The chick embryo is only fully immune-competent shortly before hatching , reducing the risk of xenograft rejection ,83,84. IHowever, developing cancer biomarkers for precision medicine demands highly translational animal models that accurately recapitulate human pathology. Moreover, in the clinics, cancer patients commonly present with multiple morbidities. Ideally, animal models should develop cancer on the background of representative comorbidities such as diabetes . Since sThe advantages and disadvantages of the described models are manifold and are presented in A successful establishment and translation of new biomarkers from a preclinical setting towards the clinical application strongly depends on the significance of the target, and the performance and explanatory power of the model in use. We are all aware of the fact that the time for developing and establishing new tracers from bench to bed is far too long. Established models and cell lines, used in the imaging community and validated for our needs for decades, provide limited insights into human disease. Experimental nuclear medicine needs to learn from and to team up with different fields of tumor biology, molecular pathology and oncology.In a clinical setting, personalized diagnosis of tumor diseases requires the ability and readiness to converge with different fields and disciplines. This need was already recognized by others and Sorace et al. proposed an idealized workflow model for radiology–pathology integration . Not onlIn the publication “Convergence—The Future of Health Report 2016”, the authors state literally that “Convergence comes as a result of the sharing of methods and ideas by chemists, physicists, computer scientists, engineers, mathematicians, and life scientists across multiple fields and industries. It is the integration of insights and approaches from historically distinct scientific and technological disciplines” (5). With this in mind, we wanted to give an overview of different methods and approaches in the fields of experimental nuclear medicine and molecular pathology to create a common understanding and facilitate future collaborations. We expect that functionally and biologically relevant biomarkers will allow for better diagnosis and monitoring of cancer. In addition, the combination of different biomarkers measured by non-invasive approaches such as molecular imaging and liquid biopsies might result in synergies that will eventually foster personalized cancer care.


Triple-negative breast cancer (TNBC) is a highly heterogeneous and aggressive cancer that has the highest mortality rate out of all breast cancer subtypes. Conventional clinical treatments targeting ER, PR, and HER2 receptors have been unsuccessful in the treatment of TNBC, which has led to various research efforts in developing new strategies to treat TNBC. Targeted molecular therapy of TNBC utilizes knowledge of key molecular signatures of TNBC that can be effectively modulated to produce a positive therapeutic response. Correspondingly, RNA-based therapeutics represent a novel tool in oncology with their ability to alter intrinsic cancer pathways that contribute to poor patient prognosis. Current RNA-based therapeutics exist as two major areas of investigation—RNA interference (RNAi) and RNA nanotherapy, where RNAi utilizes principles of gene silencing, and RNA nanotherapy utilizes RNA-derived nanoparticles to deliver chemotherapeutics to target cells. RNAi can be further classified as therapeutics utilizing either small interfering RNA (siRNA) or microRNA (miRNA). As the broader field of gene therapy has advanced significantly in recent years, so too have efforts in the development of effective RNA-based therapeutic strategies for treating aggressive cancers, including TNBC. This review will summarize key advances in targeted molecular therapy of TNBC, describing current trends in treatment using RNAi, combination therapies, and recent efforts in RNA immunotherapy, utilizing messenger RNA (mRNA) in the development of cancer vaccines. Breast cancer (BCa) is one of the most frequently diagnosed cancers in women and presents as a major public health concern worldwide. In 2018, there were an estimated 2.1 million new cases identified around the world, of which approximately 627,000 cases resulted in death . It is mGene expression profiling studies have revealed seven distinct subgroups under which TNBCs can be classified: basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal-like (M), mesenchymal stem-like (MSL), luminal androgen receptor (LAR), immunomodulatory (IM), and unstable (UNS) ,8,9. EacThe molecular classification of TNBC has progressed significantly in the past few years, paving the way for more accurate molecular characterization of TNBC tumors. The approach used above is based on the Lehmann subtype classification, which has evolved into the Burstein four subtype classification. This system divides the former six subtypes into basal-like immunosuppressed (BLIS), basal-like immuno-activated (BLIA), LAR, and MES . The BLIS subtype “expresses the immunosuppressive molecule V-set domain-containing T-cell activation inhibitor 1 (VTCN1)”, while the BLIA subtype “expresses STAT signal transduction molecules and releasing cytokines” . The BurWithin each of the Lehmann subtypes, and their respective implicated signaling pathways, there are important molecular markers present that can be targeted, offering potential therapeutic options. For example, the MSL subtype is characterized by the increased expression of VEGFR2, which can modulate VEGF activity, a major mediator of angiogenesis. Src inhibitors can be used in the inhibition of VEGFR2 tyrosine kinase activity and thus decrease tumor angiogenesis. Several promising molecular targets and their associated subtypes are included in Both the BL1 and BL2 subtypes are characterized as basal-like according to their intrinsic BCa subtype. The BL1 subtype is further characterized by mutations in genes controlling DNA-damage response, cell division, cell proliferation, and cell cycle gene expression pathways, while the BL2 subtype is characterized by having mutations in pathways regulating glycolysis, gluconeogenesis, and growth factor signaling, in addition to those mutations found in the BL1 subtype. Due to enhanced signaling activity in pathways involved in cellular proliferation, motility, and division, the tumorigenic potential of both these subtypes is increased, which in turn facilitates tumor invasion. The M and MSL subtypes, both unclassified according to their intrinsic BCa subtype, are characterized by epithelial–mesenchymal transition (EMT), where epithelial cells are transformed into invasive, migratory mesenchymal stem cells after the loss of their cell polarity and cell–cell adhesion properties . Both suAt this time, the application of targeted therapies in TNBC treatment is limited; however, promising research in the area of personalized medicine is currently underway. Targeted therapies are specific to each molecular subtype of TNBC, as they exploit knowledge about the genomic alterations that occur in each subtype to formulate individualized treatments. Specific examples of these treatments are summarized in The LAR and IM subtypes of TNBC are more unique in terms of their molecular signaling targets, wherein they are associated with upregulated genes involved in more complex pathways for androgen metabolism and immune signaling, respectively. For the LAR subtype, potential therapies can include androgen receptor (AR) antagonists that inhibit AR signaling, or using other nonsteroidal antiandrogens to inhibit cell growth. For the IM subtype, the most effective targeted therapies would inhibit immune checkpoint proteins, such as PD-1/PD-L1 and CTLA-4, which are usually over-expressed in cancers. Due to this, T cell activation is suppressed, resulting in tumor cell survival. Such immune checkpoint proteins are involved in suppressing immune responses, and so selectively inhibiting these molecular targets may lead to a strengthened immune response against cancer cells. Lastly, the UNS subtype has a characteristic upregulation of genes involved in DNA-damage response and cell proliferation signaling pathways, and so potential targeted therapies for this subtype can include inhibition of PLK1 and TTK amplification, both molecular targets involved in cell proliferation.Due to various levels of pathologic overlap between the seven molecular TNBC subtypes, there is the potential that several targeted therapies can be used to treat multiple subtypes. For example, antimitotic or cytostatic agents targeting specific proteins involved in cell proliferation signaling pathways, including PI3K and Myc, can be potentially used to treat many of the subtypes. However, despite the versatility in such therapies, there are still several intrinsic disadvantages that must be considered, principally, the potential for drug resistance of tumor cells and the lack of high specificity of these therapies. With recent developments in tumor therapeutics, however, RNA-based therapies emerged. Unlike traditional therapeutics that have a more generalized action and thus lack specificity, or have a limited range of targets, RNA-based therapies have higher specificity, a wider range of targets, and good drug properties due to their ability to inhibit a variety of genes implicated in several cellular pathways that are involved in tumor cell proliferation, motility, and survival. With the ability to “target multiple key sources of multi-gene diseases such as tumors”, RNA-based therapeutics can effectively “reduce drug resistance of tumor cells and arrest the growth of advanced-stage tumors” . Using aSimilar to the mode of action of ASOs, which intrinsically depend on the recognition of specific target mRNA sequences, other RNA-based therapies exist that specifically utilize different types of RNA to modulate certain effects on gene expression through silencing, or through the introduction of synthetic RNA. RNAi, for example, is an endogenous mechanism that primarily utilizes siRNA and miRNA for gene silencing effects, while therapies involving mRNA are primarily used for modifying protein expression in target cells and manipulating cellular phenotypes through the introduction of synthetic mRNA. The utility and effectiveness of these RNA-based therapies will be discussed in detail through the next sections, with special consideration to their application in the treatment of TNBC.The use of non-coding RNAs (ncRNAs) presents a promising therapeutic approach for the treatment of various complex disease states, among which cancer is a prime example. ncRNAs do not encode proteins but are instead involved in several important functions, including the regulation of gene expression at both the transcriptional and post-transcriptional levels . Two of ® (patisiran), is used to treat hereditary transthyretin amyloidosis (hATTR), a disease characterized by mutations in the gene encoding transthyretin and abnormal deposits (amyloids) of transthyretin protein, causing polyneuropathy and cardiomyopathy [® (givosiran), which is used to treat acute hepatic porphyria (AHP), an inherited metabolic disorder that leads to the accumulation of “neurotoxic precursors δ-aminolevulinic acid (ALA) and porphobilinogen (PBG)”, which can cause acute porphyria attacks. Givosiran is an aminolevulinate synthase 1 (ALAS1)-directed siRNA that prevents the accumulation of ALA and PGB by downregulating ALAS1 mRNA through hepatocyte-targeted delivery [siRNA-based therapeutics are actively being investigated in the field of gene therapy, wherein synthetic genetic material is inserted into host target cells to treat disease. siRNA specifically silences genes that are implicated in pathogenesis and thus produces a therapeutic effect; for example, in cancer therapy, miRNAs can suppress oncogenic mRNAs from translation and thereby inhibit tumor progression . The firmyopathy . Patisirmyopathy . Anotherdelivery .miRNA-based therapeutics has also emerged as a rapidly growing field in RNAi cancer research, though, unlike siRNA therapeutics, there have not been any approved drugs using this approach to date. Regardless, the technology has considerable potential in cancer therapeutics, due to its high versatility. Specifically, miRNA therapeutics can be used in both inhibitory and enhancing functions. Anti-miRNA therapy represents the main approach through which miRNAs are used in an inhibitory fashion, where they act as suppressors of prometastatic miRNAs. The enhancement function of miRNAs can be represented by miRNA mimicry, where synthetic miRNAs mimic the function of endogenous miRNAs involved in mRNA degradation and gene silencing. Thus, miRNA mimics can effectively enhance the function of endogenous miRNAs in gene silencing.Miravirsen (SPC3649) is an experimental drug currently in clinical testing for the treatment of hepatitis C virus (HCV) infections and is a prime example of anti-miRNA therapy. It is a modified ASO that is complementary to mature miR-122, which is a “liver-specific miRNA that is an important host factor for the life cycle of HCV” . ThroughEvidently, both miRNA and siRNA-based therapeutics are highly complex and targeted treatments that require specificity in their delivery. Currently, there are three principal delivery systems for RNAi therapeutics: polymer-based, lipid-based, and those using amorphous drug–polyelectrolyte nanoparticle complexes (or nanoplexes/nanocomplexes for short) . PatisirDendrimers are branched polymeric molecules that have also been used for siRNA delivery systems, where the most commonly used is poly(amidoamine) (PAMAM) . They arLiposomal delivery of RNAi therapeutics involves the encapsulation of nucleic acids in small artificial vesicles that can encase both hydrophilic and lipophilic drugs. These sphere-shaped vesicles consist of a membrane composed of one or more phospholipid bilayers with an internal aqueous core, and function in intracellular delivery of encapsulated material through endocytosis. Cationic liposomes have been observed to have the highest encapsulation efficiency, attributed to their specific formulation, which confers protection to nucleic acids from degradation . Cationib-2-vinyl pyridine-b-ethylene oxide) (PS-b-PVP-b-PEO), and a hydrophobic core, composed of polymers such as polypropylene glycol [Similar in structure and flexibility to liposomes, supramolecular assemblies such as micelles can also be used in RNAi-based therapeutics. Micelles encapsulate nucleic acids and drugs and are generally composed of phospholipids arranged in closed spherical monolayers, but unlike liposomes, lack an aqueous core . There ae glycol ,43. Thise glycol . PMs cane glycol ,44. Overe glycol . Examplee glycol .Cell-penetrating peptides (CPPs) are generally positively charged molecules composed of natural or synthetic short chains of linked amino acid monomers that can be used to facilitate the intracellular delivery of nanoscale particles, chemical molecules, proteins, and nucleic acids . Their pTP53. Biological features that are therapeutically targeted in various disease states are involved in the dysregulation of biological processes—in the case of patisiran to treat hATTR, for example, mutant and wild-type TTR are targeted. siRNA and miRNA can target a diverse range of molecular targets that are involved in TNBC pathogenesis, such as those implicated in dysregulated cell proliferation or motility, and growth factor signaling. Through targeted delivery of siRNA and miRNA to TNBC cells, previously dysregulated mRNAs can be inhibited from producing mutant proteins that are involved in progressing TNBC pathogenesis. Furthermore, unique characteristics of tumors can also be targeted using RNAi, such as cancer stem cells (CSCs). CSCs are located within tumors and are often implicated in several processes that are involved in cancer progression and increased aggressiveness, including metastasis, drug resistance, and disease relapse [The three NP delivery systems presented in previous sections can be used in a variety of contexts, but ultimately, the main goal for this therapeutic approach is to have specific targeting ability. RNAi can utilize these NP delivery systems to encapsulate siRNA and miRNA for targeted delivery to cells; however, specific molecular targets must first be clearly defined. As was discussed in  relapse . Several relapse . Example relapse .mRNA vaccine immunotherapy is a relatively new area of investigation within nanomedicine, which focuses on the development of personalized mRNA vaccines for the treatment of various cancers. Broadly, nucleic acid cancer vaccines contain antigens encoded by either DNA or RNA and can be further subdivided into RNA and DNA vaccines that utilize different mechanisms for therapeutic delivery. Specifically, DNA cancer vaccines consist of TSA-encoding gene(s) cloned into a bacterial plasmid . The DNACurrently, there are three general approaches for the delivery of mRNA vaccine immunotherapies: naked mRNA vaccines, encapsulated mRNA vaccines, and mRNA transfected dendritic cell (DC) vaccines. Naked mRNA vaccines are characterized as injections of free mRNA formulated only in buffer and without a carrier, such as LNPs or liposomes . AlthougThe immunogenicity of mRNA-based vaccines can be further increased through the addition of adjuvants. While naked mRNA vaccines “inherently possess self-adjuvanticity”, other molecules can be added to naked IVT mRNA to further enhance the vaccine’s ability to elicit an adaptive immune response, including poly I:C RNA, protamine, and CpG containing motifs . The tumA less-employed approach of mRNA vaccine delivery is called particle bombardment, or biolistic transfection. This method can be used to intracellularly deliver both DNA and RNA vaccines to mammalian cells through penetration of target cell membranes. This is principally achieved using IVT mRNA coated onto gold particles, which are then “accelerated toward a stopping plate by a pressurized helium pulse” . AlthougAs mentioned previously, DC transfection can be completed through electroporation, where mRNA is electrotransferred into DCs. After DCs are transfected with TAAs, they can go on to present these molecules to CD8+ T cells on MHC class I molecules. This, in turn, confers T cells with the ability to recognize these specific antigens on tumor cells and enables them to stimulate active immune responses targeting tissues expressing those specific antigens. In this way, DCs can be used to effectively target tumors in an antigen-specific manner. Similarly, total tumor mRNA can also be used to elicit host immune responses against tumors utilizing a specific profile of derived antigens. This method utilizes cancer-specific RNA, thus eliminating the need for the identification of antigens expressed by the patient’s tumor . With thMolecular targets using RNA-based immunotherapies are characteristically different from those discussed for RNAi, where instead of targeting dysregulated proteins, antigenic substances present on tumors are instead targeted to elicit host immune responses . ImmunotCombination immunotherapies can also be implemented for TAA-targeted therapies, wherein two individual therapies are used in conjunction for treatment. An example is the use of TAA-targeted mRNA nano-vaccines with CTLA-4 inhibitors . For thiDelivery of these immunotherapies will be varied but would generally consist of the subcutaneous or intravenous infusion of therapeutic mAbs, CAR-T cells and mRNA transfected DCs, and the NP, liposomal, or nanoemulsion-mediated delivery of mRNA cancer vaccines . OverallRNA-based therapies can be used in combination with traditional chemotherapeutics to enhance therapeutic efficacy and improve patient responsiveness to treatment through synergistic therapeutic effects. Several chemotherapeutics that have been used in combinatorial cancer therapy include benzethonium chloride (BZN), paclitaxel, cabazitaxel, doxorubicin, and orlistat. BZN has been used in nanocomplex formulations with Bcl-2-targeting siRNA and has demonstrated therapeutic efficacy in vivo . Hybrid Co-delivery of cabazitaxel and siRNA targeting IKBKE, an oncogene present in TNBC, using hybrid nanocomplexes also demonstrated improved therapeutic efficacy, wherein cabazitaxel enhanced the activity of the IKBKE siRNA . ExosomeCombination cancer therapies can be used to improve the efficacy and safety of current treatments utilizing small-molecule anticancer drugs, as well as amplifying the effects of RNAi. Further, combinatorial therapeutic approaches can also be used to overcome multidrug resistance (MDR), a considerable challenge in cancer treatment. Co-delivery of RNAi and anticancer drugs can produce a synergistic effect that can lead to improved treatment efficacy. As shown in With downregulation of P-gp, the concentration of the anticancer agent can be effectively increased at the target site, thus leading to greater therapeutic efficacy . As suchAs illustrated in Overall, combinatorial therapeutics presents a promising avenue for future TNBC research, as well as for other diseases. The increased efficacy of combining treatment approaches, as well as the improved quality-of-life outcomes from reduced drug toxicity, are both considerable advantages that can be gained from this modality. As discussed earlier, RNA-based interference therapies also present as a promising treatment approach in the future; however, because combinatorial therapeutics enhance the therapeutic effects of such technologies, increased research efforts must be focused on optimizing this approach for use in the clinic. The next section describes specific clinical applications of RNA-interference and combinatorial therapies for the treatment of TNBC and also delves into the ongoing challenges with the use of RNAi in TNBC molecular therapy.Preclinical development of RNA-based therapies has been ongoing for several years as novel approaches have been routinely developed and periodically introduced. Several promising preclinical studies are ongoing for TNBC treatment, many of which involve RNA-based therapies. To date, there have been several innovative therapies that have been introduced, including using novel small molecule chemotherapeutics, various immunotherapies such as those discussed earlier in The table below shows some of the current miRNAs that have been investigated in preclinical studies as potential TNBC therapies, other than those already included in There has been significant progress in developing RNA-based therapies for a wide variety of diseases, although primarily preclinically for TNBC to date. Although there are several promising early-stage studies for TNBC using RNAi and RNA immunotherapy as was described in RNA-based therapies have been increasing in recent years to treat a variety of conditions, among which TNBC has been an active area of research. To date, there have been limited clinical studies that have investigated RNA-based therapeutics for TNBC, three of which are described below in ® MUTANOME concepts, which refer to the approaches used to identify suitable molecular targets for RNA-based immunotherapy. The first concept essentially uses a patient-specific liposome complexed with mRNA of pre-identified immunogenic TAAs commonly expressed in TNBC. The second concept utilizes a two-step process, in which first, tumor-specific mutations are identified by next-generation sequencing, and second, in which on-demand RNA manufacturing is used to target neo-antigens derived from the established mutated epitopes in individual patients. In this way, RNAs are synthesized de novo to target individual tumor mutations. These two IVAC approaches can also be combined to target the entire range of antigens selectively expressed on tumors, acknowledging the heterogeneity of tumors between patients. Lastly, the third study is a current trial investigating CAR-T therapy for TNBC. Specifically, it focuses on the safety and efficacy of intratumorally administering autologous T cells in patients, redirected to target the c-Met tyrosine kinase receptor that is over-expressed in TNBC.The second study, Mutanome Engineered RNA Immunotherapy (MERIT), is an mRNA cancer vaccine trial that utilizes individualized cancer immunotherapy (IVAC), where immunogenic RNA vaccines are designed specifically for a given patient’s tumor antigen-expression profile. Two complementary strategies are used in the trial, namely the WAREHOUSE and the IVACAll the studies mentioned above present novel RNA-based therapies for the treatment of TNBC. However, upon a review of current clinical trials for TNBC treatment, it was found that most of the studies are using traditional small molecule chemotherapeutics and modern chemotherapy. Other emerging studies are investigating the efficacy of genomically directed therapies for TNBC, in turn providing personalized therapies by sequencing patient tumor total RNA or determining miRNA profiles in patients with TNBC. As such, it is evident that RNA-based therapies still comprise a relatively new and developing field that is slowly increasing in momentum for TNBC treatments. Interestingly, most of the studies that were identified in RNA-based therapies occupy a highly diverse and versatile field that can be leveraged as advanced treatments for TNBC. However, despite the significant progress described in previous sections, there remain several challenges that must be overcome before this therapeutic approach can be translated to the clinic. In DC vaccines are also a novel RNA-based therapeutic that can be implemented for use in TNBC treatment. As was discussed in Finally, combinatorial therapeutics, utilizing both RNAi and traditional chemotherapeutics, remain an active area of investigation. This field was developed in response to the various limitations that exist with RNAi monotherapy, including their restriction to mainly indirect actions involved in gene regulation, concerns about bioavailability, and several others discussed above. Combination therapies effectively address these limitations by improving the therapeutic potential of RNAi, as well as mitigating many of the disadvantages that exist with current chemotherapeutic small-molecule drugs. For example, using RNAi along with small-molecule drugs can help overcome MDR by downregulating MDR proteins, such as ABC transporters. Secondly, using both therapeutics enables for a stronger therapeutic effect due to synergism, and thirdly, many of the toxicities and associated side effects that occur with the use of chemotherapeutics can be prevented due to a lower dose of the small-molecule drug being required. However, using combination therapies also has associated disadvantages, including the potential for novel adverse reactions to develop with concurrent use of the two disparate therapeutic approaches. Furthermore, combination therapies are also considerably costly to implement, and so their long-term application may not be feasible. Ultimately, with all the therapeutic approaches described, the costs of the therapies must be weighed with their benefits, and if it is seen that their benefits far outweigh their costs, then it would be prudent to translate those therapies into the clinic. In the case of combination therapies, because there are numerous benefits associated with their use, such therapies will likely be seen in the clinic more readily. This is the same for the other treatment modalities discussed, which though are lacking in certain areas, with additional research and modifications, have the potential to be implemented in the clinic alongside combination therapies for future applications in TNBC treatment.This review discussed various targeted therapies available for TNBC, among which RNAi, immunotherapy, and combination therapies were present as the promising approaches. All these approaches have the potential to be implemented in TNBC molecular therapy and translated clinically, though understandably, this will take time. Certainly, in targeting various unique pathological distinctions in TNBC, these RNA-based poly- and monotherapies present novel approaches for treating TNBC and are promising for future use. The notable success of mRNA vaccines in the management of the COVID-19 pandemic speaks both to the efficacy and the considerable adaptability of RNA-based therapies, which will likely experience a strong resurgence in the coming years for future cancer therapies ,122. AltIn summary, targeted RNA-based molecular therapy of TNBC may be effectively achieved through various modalities, including RNAi, combination therapy, and vaccine immunotherapy. Of those discussed, immunotherapies and combination therapies represent innovative therapeutic approaches with great potential given their substantial recent advances. However, based on current clinical trials, combination therapies, compared to stand-alone immunotherapies, will likely be incorporated into TNBC treatment paradigms sooner in the near-term. Alternatively, non-RNA-based treatments, such as immunochemotherapy or radioimmunotherapy, are also both potential avenues of research that have not yet been studied as extensively for TNBC treatment. Ultimately, however, through additional research efforts devoted to addressing their key limitations, all the RNA-based therapies reviewed possess significant potential for future use in TNBC molecular therapy.


Heparin and its derivatives are saving thousands of human lives annually, by successfully preventing and treating thromboembolic events. Although the mode of action during anticoagulation is well studied, their influence on cell behavior is not fully understood as is the risk of bleeding and other side effects. New applications in regenerative medicine have evolved supporting production of cell-based therapeutics or as a substrate for creating functionalized matrices in biotechnology. The currently resurgent interest in heparins is related to the expected combined anti-inflammatory, anti-thrombotic and anti-viral action against COVID-19. Based on a concise summary of key biochemical and clinical data, this review summarizes the impact for manufacturing and application of cell therapeutics and highlights the need for discriminating the different heparins. The discovery of heparin more than one hundred years ago happened as an unexpected coincidence. At the beginning of the 20th century, William Howell and co-workers were studying a pro-coagulant ‘thromboplastic substance’  consistiHeparin is a linear, unbranched and highly sulfated polysaccharide belonging to the family of glycosaminoglycans (GAGs) ,22. BaseThe anticoagulant effect of heparins is in part mediated by a unique pentasaccharide sequence binding with high affinity to antithrombin . This inCommercially available UFH is isolated from porcine intestinal mucosa or lung and intestine from cattle, the only sources for industrial production. As outlined recently , the comUFH  has several advantages including short onset of action, no placenta passage, feasibility for patients with renal failure, neutralization by protamine and low price. Nevertheless, the limitations predominate, particularly the need for parenteral administration and dose monitoring, due to variable bioavailability. In clinical practice, the individual dose is adjusted, e.g., six-hourly, usually according to the activated partial thromboplastin time (aPTT) and the anti-FXa activity, both poorly reflecting the anticoagulant effect . As UFH LMWHs are produced from UFH by chemical and enzymatic depolymerisation, yielding smaller polysaccharide fragments (12–22 monosaccharide units with an average molecular weight of 5000 Da). Compared to an anti-FXa/anti-thrombin activity ratio of one for UFH, for LMWHs this ratio is between two and five . Due to ®) being the first and only synthetic clinically approved selective FXa-inhibitor [As just one third of individual heparin molecules show anticoagulant properties, further attempts have been made to biochemically synthesize only ULMWHs or pentasaccharides, as fondaparinux , due to diminished thrombin formation. Platelets are essential contributors of cancer-associated thromboembolism but can also nurture tumor growth and metastasis, as reviewed comprehensively . These cFurther potential non-anticoagulant anti-cancer effects of heparin were already described in 1957, demonstrating an inhibitory effect of UFH on ascites tumors in mice . FurtherThe most common adverse effect of heparins is bleeding. The incidence of major bleeding complications ranged from 2% with LMWHs to more than 5% with intravenous UFH . The indA frequently observed transient and mild decrease in platelets due to a nonimmune-mediated effect of heparins is termed type I heparin-induced thrombocytopenia (HIT). The massive platelet drop on days 3 to 5 of heparin therapy is a rare but potentially life-threatening side effect. The characteristic symptoms of a more distinct thrombocytopenia and arterial embolism, now considered type II HIT, were first described by Weismann and Tobin in 1958 . The mecA variety of stimuli regulates the extracellular matrix (ECM) conformation, including mechanical forces and different ligands . During Heparin and heparan sulfate significantly regulated genes involved in cell adhesion and proliferation in human bone marrow-derived stromal cells in a donor-dependent manner ,82. UFH The effect of UFH supporting long-term propagation of endothelial cells is well-known . It playThe effect of heparins on cell proliferation in vitro can be either growth promoting or inhibiting. It appeared to be mandatory for efficient proliferation of endothelial cells and their progenitors ,109. StrGAGs are also tightly linked to developmental and differentiation processes. In animal models, proteoglycans were identified as important modulators of protein gradient formation and signal transduction ,114. UFHEndothelial cell culture as a prerequisite for studying molecular mechanisms of vascular biology and regeneration was established half a century ago ,123. AddSolid organs and different progenitor cell types can be transplanted successfully due to human leukocyte antigen matching and pharmacologic immune suppression strategies. After liver cell transplantation, an initially unexplained substantial cell loss after application was discovered through attentive observation ,138. TheIn order to prevent thrombotic complications after transfusion, several animal studies and subsequent clinical trials were efficiently using UFH as a pretreatment ,151,152 A growing number of biotechnology applications is using heparins to support the production of specific cell-based therapeutics. The precise nature of the heparins (UFH or LMWH) was commonly not disclosed. The most frequently used heparin-based biomaterials include heparin-functionalized surfaces , heparin-based hydrogels , and heparin-containing nanoparticles, micelles or so-called coacervates, spontaneous aggregates of amphiphilic molecules . HeparinHeparins were successfully tested for tissue engineering (not covered in this review) and improving the production of cell therapeutics as a key component of diverse biomaterials. These functions, which are quite different to the anticoagulant properties of soluble heparin, might be explained in part also by the immobilization and associated modifications, which are supposed to affect heparin’s functionality in addition to the multiplicity of mechanistic explanations discussed in detail above.A century after heparin discovery, mainly LMWHs and synthetic pentasaccharides are used in daily clinical practice together with oral anticoagulants to prevent and cure thromboembolic events . However