Exosomes are cell-secreted nanovesicles that naturally contain biomolecular cargoes such as lipids, proteins, and nucleic acids. Exosomes mediate intercellular communication, enabling the transfer biological signals from the donor cells to the recipient cells. Recently, exosomes are emerging as promising drug delivery vehicles due to their strong stability in blood circulation, high biocompatibility, low immunogenicity, and natural targeting ability. In particular, exosomes derived from specific types of cells can carry endogenous signaling molecules with therapeutic potential for cancer treatment, thus presenting a significant impact on targeted drug delivery and therapy. Furthermore, exosomes can be engineered to display targeting moieties on their surface or to load additional therapeutic agents. Therefore, a comprehensive understanding of exosome biogenesis and the development of efficient exosome engineering techniques will provide new avenues to establish convincing clinical therapeutic strategies based on exosomes. This review focuses on the therapeutic applications of exosomes derived from various cells and the exosome engineering technologies that enable the accurate delivery of various types of cargoes to target cells for cancer therapy. Extracellular vesicles (EV) are one of the means of intercellular communication between adjacent and distant cells. EVs refer to all membrane vesicles that are naturally released from cells and are classified based on their biogenesis pathway, function, size, etc. . EVs areAmong them, exosomes play essential roles as communication mediators between cells and contain many important biomolecules such as proteins, nucleic acids, and lipids . These cThe hurdles of some artificial nanoparticles, such as immune recognition , inflammIn this review, we aim to provide a comprehensive understanding of exosomes, including their biogenesis, function, therapeutic applications for cancer treatment, as well as various engineering techniques in drug delivery.The exosome is a small vesicle of endocytic origin with a diameter of 40–150 nm and contains many types of biomolecules such as proteins and nucleic acids . The bioEven exosomes derived from the same cell are heterogeneous in their size and cargo. However, some cargoes are partially common in exosomes derived from various origins. . AccordiAccording to current studies, the mechanism by which exosome signals are transferred to recipient cells can be largely defined in three methods: receptor interaction, direct membrane fusion, and endocytosis/phagocytosis. For example, Cosseti et al. found that interferon-γ (IFN-γ) binds to exosome-associated IFN-γ receptor 1 to form a complex, which in turn activates signaling transduction through this complex in neural stem/precursor cells . In addiExosomes play crucial roles in intercellular communication by delivering biomolecules. Exosome-derived biomolecules secreted by donor cells could effectively change the biological response of recipient cells. Zhang et al. found that the exosome with a significant amount of miRNA-150 secreted by human monocytic leukemia cell line (THP-1) could contribute to target cell migration through the inhibition of target gene expression . In anotIn our body, natural-killer (NK) cells are responsible for immune surveillance and serve as the first-line defense in regulating cancer growth and metastasis. NK cell-derived exosomes endow cytotoxic activity to tumor cells by carrying killer proteins together with typical NK and exosome markers .Similar to NK cells, macrophages play an important role in innate host defense and in killing tumor cells by producing reactive oxygen/nitrogen species and pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). Macrophages are broadly classified into M1 or M2 types, and tumor-associated macrophages (TAMs) in the tumor microenvironment are known to exhibit an M2-like phenotype. These TAM-derived exosomes induce immune suppression for tumor progression by mediating cell-to-cell communication with other immune cells such as T cells .+ tumor-associated macrophages population that inhibits CD8+ T-cell function [Tumor-derived exosomes (TEXs) released from malignant cells alter the physiology of macrophages , reduce function . The stufunction . AdditioTherefore, the application of TEX as biomarkers of cancer therapy is becoming increasingly developed due to their function in TME. The role of TEXs as a biomarker has been studied in various recent studies. One study demonstrated that TEXs in non-small cell lung cancer (NSCLC) could also be used as therapeutic biomarkers as potential cancer diagnostic indicators . The othThese findings including various effects of TEXs on cancer pathogenesis support that they may become potential therapeutic agents and tools for cancer therapy.As mentioned previously, cell-derived exosomes have shown potential therapeutic effects through cargo contents and functions received from donor cells. This potential can be maximized by engineering the exosome as a natural nanoplatform for drug delivery. With the advancement of nanotechnology, nanomedicine has made an important contribution to improving the loading capacity, biodistribution, and target accumulation of therapeutic molecules . The advRecently, many papers have reported the loading of a variety of therapeutic molecules including chemical drugs, proteins, and nucleic acids into exosomes. As a nanoplatform, the inner space of exosomes can be the room for drug loading. In addition, as previously mentioned, exosomes have internal contents, including proteins and miRNAs, inherited from donor cells. The use of exosomes as a drug delivery vehicle can be mainly discussed in four ways: (1) co-incubation, (2) membrane permeability enhancement, (3) cytoplasmic abundance in donor cells, and (4) selective encapsulation in exosome.Co-incubation with exosomes and therapeutic molecules is the simplest encapsulation method using the space of the membrane lipid bilayer. The small hydrophobic molecules such as curcumin  and doxoTo overcome the limitations of the co-incubation method, new approaches have been proposed to improve exosomal membrane permeability through physical or chemical stimulation. These stimulations are commonly used methods to improve intracellular uptake or induce the membrane deformation of cells, and they can also be applied to exosomes with the same membrane structure. Membrane permeability can be improved by the generation of pores on the exosomal membranes, disruption of membrane integrity, or transfection with positively charged agents. While these approaches ideally increase drug loading efficiency, the aggregation of exosomal membranes can be induced by these stimulations.As pore generating stimuli, electroporation and saponin permeabilizer have been studied for membrane permeability enhancement. Electroporation is a method of incubating exosomes with the desired therapeutic agents and exposing them to a repetitive electric field of a certain voltage to make a pore in the exosomal membranes to encapsulate therapeutic agents . This meTo disrupt exosomal membrane integrity, sonication has been explored for drug encapsulation into exosomes. The membrane deformation through the ultrasonic homogenizer probe causes the drugs to diffuse into exosomes. Kim et al. demonstrated that exosomal membrane microviscosity was decreased by sonication . As the Lastly, therapeutic nucleic acids can be loaded into the exosomes with transfection agents. Shyam et al. showed siRNA encapsulation in exosomes using a commercial transfection agent, lipofectamine . The RADOne of the main approaches for drug encapsulation into exosomes is the overexpression of the drug in donor cells. Therapeutic molecules including chemicals, nucleic acids, proteins, and nanoparticles in the cytoplasm can be loaded in the exosomes due to their abundance. Pascucci et al. treated a low dose of paclitaxel to mesenchymal stroma cells for 24 h . After sCollectively, the method inducing the cytoplasmic abundance in donor cells showed a facile encapsulation of various therapeutic molecules with a simple mechanism. However, note that the toxicity of donor cells and inefficient drug loading limit its broad applications.To overcome the limitations of the method for cytoplasmic abundance in donor cells, a method of selective drug encapsulation into donor cells was explored. Particularly, unlike chemical drugs, large molecules including proteins and nucleic acids are hardly passively encapsulated into the exosome lumen. However, therapeutic molecules can be selectively loaded into exosomes by targeting the constituent proteins of exosomes involved in exosome biogenesis and packaging . The interaction between the constituent protein of the exosome and the therapeutic molecule leads to a specific encapsulation of the molecule into the exosome.Di Bonito reported a fusion protein which is the Nef exosome-anchoring protein fused with human papillomavirus E7 (Nef/E7) for exosome-based immunization. . The NefFor the selective encapsulation of nucleic acids into exosomes, exosome-enriched RNAs (eRNAs) have been studied with the distinguishing of enriched sequences in the exosomes . The eRNThese proteins such as MVP, GW182, AGO2, and Myoferlin may be be a tool for loading specific nucleic acids into exosomes. More detailed and accurate bioinformatics analysis will be needed to discover new targets for encapsulation into exosomes.Although exosomes have promising therapeutic potential, there are several challenges that must be overcome before transitioning from the laboratory stage to clinical use. Some of these challenges include the low yield of exosomes and non-standardized isolation and purification methods. In order to overcome these hurdles, biomimetic exosomes that can be generated in all cell types with characteristics similar to exosomes for an alternative therapeutic drug modality have been developed and studied .The exosomal membranes, the surface of the nanoplatform, is exposed to the external environment. In the field of nanomedicine, surfaces play an important role in therapeutic applications including extended circulation and specific activity targeting. Exosomes have the same composition and formation as the donor cell membrane. Thus, they express an intrinsic membrane pattern that includes lipid composition, adhesion molecules, specific ligands, and membrane-associated enzymes. The exosome engineering with modification of their membrane mainly focused on adjusting this pattern to increase the therapeutic effect.Exosomal membranes can be modified with membrane proteins expressed by transfection with plasmid DNAs or mRNAs in donor cells. Since membrane proteins on the cellular membranes are inherited to exosomes with the native forms associated with lipid bilayers, the exosome is a unique platform for maximizing the membrane protein activity . With meAlternatively, rather than using molecular cell biology approaches to enable downstream modifications of exosome surface macromolecules, exosomal membranes can be modified by post-insertion methods. The post-isolation modification techniques enable the functionalization of exosome surfaces with specific moieties to improve targeting ability and biodistribution and allow tracking in vivo and in vitro. The exosome surface can be essentially modified by (i) covalent interaction between functionalizing molecules or the chemical linker and the amine groups, which are reactive functional units widely expressed on exosomes’ surfaces, and (ii) non-covalent interaction based on lipid-conjugated compounds post-insertion into exosomal membranes. Recent study has reported that polyethylene glycol (PEG)-modified exosomes were able to avoid opsonization and extend the circulation half-life of exosomes. These exosomes were modified through the incorporation of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine into the lipid layer of the exosome. Furthermore, for targeting lymph nodes, the PEG’s distal end was functionalized with amine groups followed by conjugating with mannose-isothiocyanate . Kim et In this way, the development of exosomal membrane engineering technology can maximize the efficacy of exosomes through an improved circulation time and targeting ability of exosomes.With advantages such as high biocompatibility, increased circulating stability, low immunogenicity, and toxicity, exosomes can be used as attractive therapeutic delivery vehicles for delivering functional genetic material to the targeted cells. A number of studies have been conducted to show that a variety of cell-derived exosomes can be used as therapeutic agents for cancer therapy .TEXs are commonly used with chemotherapeutics to enhance the efficacy of cancer treatment because it is easy to deliver to tumor cells through the surface proteins of TEX. TEXs loaded with doxorubicin through electroporation showed a considerable inhibition of tumor growth than the free doxorubicin group and also showed the cardioprotective effect by restricting the uptake to myocardial endothelial cells .Tumor cells express antigens that can be recognized by cytotoxic T lymphocytes (CTLs). TEXs are also able to present tumor antigens, indicating that TEXs can be modified for cancer immunotherapy. Previous studies have shown that dendritic cells (DCs) can take up TEXs containing donor antigens and induce antigen-specific CTL responses in vitro or in vivo. As a result of the stronger CTL activation and faster DC uptake rate, the tumor-suppression effect of TEX-loaded DCs was significantly higher than that of tumor lysate-loaded DCs in a myeloid leukemia WEHI-3B-bearing mouse model . MeanwhiRecent research has confirmed that the M2 isoform of pyruvate kinase plays a key role in catalyzing glycolysis. Circular RNA identified as a sponge of miR-122 targeting PKM2 was loaded onto TEXs and delivered to drug-resistant colorectal cancer. TEXs inhibited glycolysis by reprogramming tumor metabolism, weakened drug resistance, and showed excellent anticancer effects in in vitro and in vitro experiments . TEX, whThe current study of stem cell-derived exosomes mostly uses MSCs because of their ex vivo expansion capacity and ethical acceptability . AlthougFor example, exosomes derived from mouse bone marrow MSCs (BM-MSCs) have reported to inhibit angiogenesis in breast cancer (4T1) by downregulating VEGF expression in vivo and in vitro . MiR-16,Recent studies have shown that immune cells such as DCs, NK cells, T cells, and macrophages can secrete exosomes that affect many physiological and pathological processes . Thus, i+ T cells, increasing IL-2 expression, and decreasing regulatory T cells [DCs-derived exosomes (Dexs), the professional antigen-presenting cells necessary to maintain innate and adaptive immunity, have been extensively studied . DCs are T cells .Similar to Dexs, NK-derived exosomes are also highly promising antitumor therapeutic candidates. NK-derived exosomes are known to contain a typical NK marker such as CD56 and cytotoxic molecules including granulysin, granzyme, perforin, and Fas ligand . One of + T cells can induce the apoptosis of mesenchymal stem cells in tumor-bearing mice, thereby attenuating tumor growth [While DC-derived exosomes have been extensively studied, there are few studies on exosomes derived from T cells. T cell-derived exosomes, similar to their parental cells, are mainly associated with antiviral and antitumor responses, and they carry the TCR/CD3 complex from activated T cells . One stur growth . Chimerir growth . Thus, cr growth .Macrophage-derived exosomes can also regulate the immune response and induce macrophage polarization. From the results of analyzing the mRNA content of activated macrophages and exosomes derived from these cells, it can be inferred that the state of macrophages was reflected in the secreted exosomes . One stuIn addition to the above-mentioned cells, therapeutic applications using exosomes derived from various cells have been studied. The HEK 293T cell line is often used to deliver exogenous therapeutic proteins, nucleic acids, and drugs. For example, HEK293T cells-derived exosomes-loaded super-repressor IκB inhibits the inflammatory responses by reducing the secretion of the NF-κB-mediated pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 contributing to cancer development . In anotFurthermore, another study identified that the encapsulation of doxorubicin in HEK293T-drived exosomes has potential properties for the effective delivery into target cells. The use of engineering exosomes-loaded doxorubicin via electroporation enhances the cellular uptake efficiency and the therapeutic effect compared to other doxorubicin formulations . In the Recently, adipocyte-derived exosomes that are involved in the response and regulation of metabolic states are emerging as promising therapeutic agents. One study reported that 3T3-L1 adipocyte-derived exosomes could enhance the expression of MMP3 in cancer cells that alleviated lung cancer metastasis by activating MMP9 . On the The outstanding advantage of exosomes as a drug delivery vehicle lies in their biological origin, which is related to their biocompatibility as a natural nanoplatform. In addition, exosomes have excellent performance in prolonging blood circulation time, increasing tumor targeting, and tumor inhibition. Notably, the use of autologous exosomes has suggested a promising therapy as a personalized nanomedicine . HoweverOne of the major challenges to realizing exosomes-based treatments is the low productivity of exosomes. The methods for exosome isolation from the cultured medium are based on ultracentrifuge or filtration at laboratory scales. The yield of exosomes obtained from these methods may vary slightly depending on the type of donor cells but is generally too low. The yield of exosomes is typically less than 1 µg of exosomal protein per 1 mL of culture medium, while the functional dose of exosomes is approximately 10–100 µg exosomal protein per mouse in most studies ,116,117.Recent reports suggest that exosomes derived from the same parent cell may have different molecular composition ,123,124.In terms of engineering, therapeutic molecules including chemical drugs, nucleic acids, and proteins have been loaded into exosomes. In the process of the engineering, exosomes and donor cells may be affected on exosome content or protein composition. Especially, engineering using physical or chemical stimulation may induce changes in the size and surface potential of the exosome. This may affect the bioactivity and therapeutic efficacy of the exosome. Therefore, exosome engineering should be developed with little impact on the bioactivity, morphology, and composition of exosomes. In addition, similar with content analysis, the developments need to be linked to standards for the evaluation of encapsulation methods.Despite the challenges, the exosomes have shown a great potential in clinical trials for cancer therapy  and woul


Traditional anticancer therapies can cause serious side effects in clinical treatment due to their nonspecific of tumor cells. Aptamers, also termed as 'chemical antibodies', are short DNA or RNA oligonucleotides selected from the synthetic large random single-strand oligonucleotide library by systematic evolution of ligands by exponential enrichment (SELEX) to bind to lots of different targets, such as proteins or nucleic acid structures. Aptamers have good affinities and high specificity with target molecules, thus may be able to act as drugs themselves to directly inhibit the proliferation of tumor cells, or own great potentialities in the targeted drug delivery systems which can be used in tumor diagnosis and target specific tumor cells, thereby minimizing the toxicity to normal cells. Here we review the unique properties of aptamer represents a great opportunity when applied to the rapidly developing fields of biotechnology and discuss the recent developments in the use of aptamers as powerful tools for analytic, diagnostic and therapeutic applications for cancer. In recent years, many progresses have been made in treating diseases including cancer due to rapid development in many aspects. Globally, the high mortality rate of malignant tumors still critically endanger people's lives and health due to challenges arising in the clinical management and diagnosis of cancer Aptamers are single stranded DNA, RNA, or altered nucleic acids sequences with a strong affinity for specific target binding generated by the systematic evolution of ligands by exponential enrichment (SELEX) technology As synthetic molecules, aptamers modifications occur regularly and toward a specific purpose. Up to now, lots of modifications to aptamers have been applied to make them more versatile, such as modified with biotin, florescent dyes or radionuclides. In generally, the physical and biochemical properties of aptamer influence its biodistribution or pharmacokinetics Nano-sized particles or materials are made of biocompatible and biodegradable materials. It can efficiently deliver proteins, peptides or nucleic acids into specific cells Table 1). Studies have shown that the dissociation constant of some RNA aptamer-target complexes can reach picomolar levels, showing that the aptamers have stronger affinity for binding targets than antibodies. Aptamers have high specificity, and can strictly recognize the difference between a target molecule and non-target molecules or even a group and different amino acids The term “aptamer”, derived from the Latin 'aptus' meaning 'fit', was first reported in 1990 by Ellington et al. 13-1016 different sequences in vitro by molecular biology technology, and then incubate the random library with the target to isolate the binding sequence. After that, the PCR amplification product can be used for the next round of screening, which can be enriched after several rounds of screening. A nucleic acid library with high affinity to the target is collected, and a specific aptamer sequence can be obtained after cloning and sequencing Figure 1). Although the conventional SELEX can effectively screen aptamers in vitro, a typical SELEX process can take weeks or even months. In recent years, with the emergence of various new, efficient and high-throughput screening methods, including automation technology, capillary electrophoresis technology, microfluidic chip technology, nanotechnology, high-throughput sequencing technology, etc., there has been significant development both in in screening objects and screening efficiency Aptamers are selected by SELEX technology, which begins with a chemically-synthesized oligonucleotide library that contains 10Table 2). The cell-SELEX screening process is similar to traditional SELEX, including incubation, isolation, and amplification. Unlike traditional SELEX targeting individual target molecules, the target of cell-SELEX is intact living cells, retaining the natural conformation of cell surface proteins, and the screened aptamers are more suitable for biological applications. Since there is no need to first detect the molecular markers on the cell surface, it brings great convenience to the aptamer selection process In addition, cancer cells are characterized by cell surface molecules that are over-expressed or altered due to multiple oncogenic mutations, so scientists have further developed cell-SELEX aptamer screening technology Figure 2). Specific modifications facilitate the delivery aptamers into target cells with precised specificity. Some modifications of aptamers by functional optimization, multimerization or truncation have been shown to enhance the stability and binding efficacy. Various modification designs, conjugation strategies and linkage approaches are prevalent in aptamer technology Considering the wild-type aptamer molecules have very short half-life due to the clearance from the body by kidney and nuclease-mediated degradation, which limited their application under physiological conditions. Therefore, a number of modified aptamers through biochemistry approaches have been used to overcome aptamer's instability and optimize the pharmacokinetic and pharmacodynamic properties of aptamer In recent years, chimeric aptamers have become an insight because of their greater stability and nuclease resistance. Chimerization of aptamers refers to its combination with another aptamer, siRNA, protein, enzyme, biomacromolecules, drug, imaging agents or dyes With the progress of research in this field, it is very clear that chimeric aptamers are not only highly stable and efficient but are also able to deliver drugs. For instance, The anticancer aptamers chimera systems are able to specifically bind to tumor cells and deliver their drugs to the target cells Most of the traditional anti-tumor drugs are not selective and have caused serious toxic and side effects in clinical treatment Currently, a variety of aptamers targeting tumor cells have been screened by SELEX; for example, A10, anti-prostate-specific membrane antigen (PSMA) aptamer In recent years, aptasensors, a biosensors that use aptamers as bio-receptors, have been attracted noticeable attention for cancer biomarker detection Aptamer-based optical sensors had made great improvements in a variety of fields such as life science, medicine and environmental monitoring Aptamer-based electrochemical sensors, as an important sub-group of sensors, are highly attractive and applicable for diagnosis of cancer and use in point-of-care tools, in addition to multi-analyte detection in vivo imaging, etc. in vivo imaging.Cancer is the second leading cause of death worldwide 2O2), Pt-Au nanoparticles quickly catalyzed the colorimetric reaction and transmit a sensitive signal. The system has a detection limit of 10 cells/mL and a linear range of 10 to 105 cells/mL Sensitive detection of cancer cells plays an important role in in cancer diagnosis and prognosis In vivo imaging technologies for tumor diagnosis include luminescence imaging, computed tomography (CT), magnetic resonance imaging (MRI), radionuclide-based positron emission tomography (PET), and single photon emission computed tomography (SPECT). For example, IRD800CW labeled CD30 aptamer is used for in vivo imaging of lymphoma in vivo imaging of pancreatic cancer in vivo imaging of gliomas Molecular imaging is of great significance in disease detection, surveillance and prognosis Epidermal adhesion molecule (EpCAM) aptamer conjugated with magnetic nanomaterials for MRI imaging of gastric cancer In recent years, aptamers have been widely used in IHC analysis of tumor tissues, and have shown some application characteristics superior to antibodies. Zu's group performed IHC analysis of formalin-fixed-paraffin-embedded lymphoma samples and found that the CD30 aptamer showed almost the same staining pattern as the CD30 antibody, but its reaction conditions were simpler than antibody. Such as antigen retrieval temperature is 37 °C (CD30 antibody is 95 °C), incubation time is 20 minutes (CD30 antibody is 90 minutes). In addition, unlike antibodies, aptamers do not cause non-specific staining of necrotic areas in tissue samples when applied to IHC Figure 3).At present, many chemical drugs can effectively kill cancer cells, but also destroy normal tissue cells and cause serious adverse reactions. Therefore, targeted drug delivery is the key to current tumor treatment. Aptamers have become a new direction for tumor-targeted drug therapy and an ideal tool for therapeutic applications due to their unique physical and chemical properties Table 3). For example, the first aptamer targeted to human VEGF for the treatment of age-related macular degeneration was approved by food and drug administration (FDA) in 2004 in vitroWith development of aptamers selected from Cell-SELEX, an increasing number of aptamers are capable for use as therapeutic drugs for disease  models have also been successfully established in recent years and are undergoing extensive preclinical evaluation Gene drugs such as siRNA and microRNA also have important clinical application value. However, the lack of targeting and inability to effectively enter tumor cells has greatly reduced the feasibility of its clinical application. Conjugating with aptamers can increase many application advantages of gene drugs, such as making it obtain tumor targeting ability, and improving the efficiency of entering cells by receptor-mediated internalization pathways. In recent years, many aptamer-conjugated gene drug models have been established, and extensive preclinical evaluations have confirmed their feasibility for tumor treatment, such as PSMA aptamer conjugated with Plk1 or Bcl2 siRNA for prostate cancer treatment in vivo and in vitro studies. In addition to covalent coupling, for some aptamers containing consecutive paired GC/CG sequences in a three-dimensional structure, anthracycline antitumor drugs (such as Dox) can be incorporated by non-covalent binding in the GC/CG structure of aptamers, forming a simple and effective AptDC model, such as epidermal adhesion molecule (EpCAM) aptamer coupled with doxorubicin for the treatment of retinoblastoma Chemotherapy is primary approach for cancer treatment. However, the shortcomings of chemotherapeutic drugs are inevitable owing to serious toxic side effects, such as their poor water solubility, nonspecific distribution and systemic toxicity greatly reduce delivery efficiency, which limits their use in clinic. Up to now, targeted chemotherapy is vital to avoid its side effects and enhance its therapeutic efficiency Table 4).Due to the small molecular weight of the aptamers, the half-life in the blood circulation is short, which seriously affects the efficacy. Nanomaterials with a certain range of diameters can target to tumor cells via the enhanced permeability and retention (EPR) effect. However, this way of increasing drug enrichment through the EPR effect is still a “passive” targeting mode, which is susceptible to some factors such as structural differences in new blood vessels, blood pressure, and the pathological type and location of tumors, resulting in significant individual effects difference Due to the special photothermal effect of gold-silver nanoparticles, Huang et al. conjugated it with the aptamer sgc8c that specifically recognizes CCCRF-CEM cells to form an Apt-NPs complex, which can be used for photothermal treatment. The results show that the Apt-NPs complex can not only target tumor cells, but the temperature of AuNPs will continue to increase under near-infrared light to kill tumor cells, but it will not cause damage to normal cells , reduced required laser exposure, and greatly reduce the amount of laser irradiation required In vitro, the drug co-delivery system can specifically target PSMA-expressing prostate cancer cells and show significantly higher tumor killing activity than single drugs Zhang et al. established an aptamer-targeted nanodrug co-delivery system. In this system, a PSMA aptamer containing a GC/CG repeat is used to assemble the hydrophilic drug doxorubicin and a polylactic acid-glycolic acid copolymer (PLGA) is used to encapsulate the hydrophobic drug docetaxel. After the drug assembly is completed, the aptamer and PLGA nanoparticles are coupled by PEG molecules. Pala et al. constructed dextran-encapsulated magnetic nanomaterials, which were combined with DNA aptamer that specifically recognized HER2 for magnetic hyperthermia of tumor cells. The results show that this system can highly specifically target SK-BR3 cells over-expressed by HER2, and 50% of SK-BR3 cells can be killed by magnetic hyperthermia. The therapeutic effect is 90 times that of magnetic nanomaterials alone. Moreover, the survival rate of HER2-negative U-87MG cells is close to 100%, which greatly reduces the dose of magnetic nanomaterials required for traditional magnetic hyperthermia and further reduces the side effects of treatment Figure 4). They provide a similar or even superior ability to that of the corresponding monoclonal antibody, and their superior targeted delivery ability confers on them less off-target side effects As a relatively novel method for cancer therapy, cancer immunotherapy has become increased attention due to its high specificity and low side effects, even rivaling other standard treatments Aptamer, a novel specific combining tool to various types of target, has attracted an increasing attention for cancer diagnosis and therapy. In this review, we review recent advances in this promising field of aptamer, including the screen of aptamer by SELEX process, modification of aptamers and applications of aptamenr for biosensing, bioimaging and therapy in cancer. Although aptamers have made great progress in tumor application research, they still need to continue to improve of drug loading rate, targeting efficiency, circulation time, and affinity, etc. The combination of aptamers and drugs and the modification of nanocarriers by aptamers still need to be improved. With the continuous advancement of SELEX technology and chemical modification methods, we believe that aptamers will definitely play a more and more important role in future oncology applications.


Non-alcoholic fatty liver disease (NAFLD) is comprised of a spectrum of liver injury ranging from excess fat accumulation in the liver (steatosis), to steatohepatitis (NASH), to its end stage of cirrhosis. A hallmark of NAFLD progression is the decline in function of hepatic mitochondria, although the mechanisms remain unresolved. Given the important role endothelial nitric oxide synthase (eNOS) plays in mitochondrial dynamics in other tissues, it has emerged as a potential mediator of maintaining mitochondrial function in the liver. In this mini review, we summarize the most relevant findings that extends current understanding of eNOS as a regulator of mitochondrial biogenesis, and identifies a potential additional role in mitochondrial turnover and attenuating inflammation during NAFLD development and progression. Non-alcoholic fatty liver disease (NAFLD) is a progressive disease of the liver that ranges on a wide pathological spectrum from hepatic steatosis to a more severe non-alcoholic steatohepatitis (NASH) phenotype, which can progress to fibrosis and cirrhosis . Numerou2O2 emission, and reduced anti-oxidant capacity , three inhibitory sites , and two sites for which the influence on eNOS activity is undetermined  . Of partogenesis . Interesogenesis  or metfoogenesis  does notogenesis . This leogenesis .Mounting evidence from whole body eNOS KO models demonstrate its importance for metabolic health. Mice lacking eNOS have elevated FFAs and triglycerides, as well as reduced mitochondrial content and beta-oxidation in skeletal muscle . AdditioLeprdb/db) mice crossed with eNOS KO mice exhibit elevated hepatic steatosis compared with Leprdb/db , in hyperphagic OLETF rats  that is present on the inner mitochondrial membrane. As there is no fourth NOS gene in mammalian genomes, mtNOS is thought to be a variant of one of the three known NOS isoforms. Initial reports identified eNOS as the mtNOS in skeletal muscle, brain, and liver . However2 concentrations. These data indicate that the rate of O2 consumption at a given [O2] is inversely proportional to [NO]. In addition, NO may influence other aspects of mitochondrial bioenergetics, including attenuation of complex I and III, which are known sites for ROS production through S-nitrosylation  derived NO is its well-characterized interaction with COX in the mitochondrial ETC. NO inhibits COX via both competitive and non-competitive sites . Importasylation , and NO-sylation . Thus, csylation . Taking The link between eNOS derived NO and its regulation of mitochondrial function in other tissues is well established. In a seminal paper by While there is clear evidence for a role of eNOS in the process of mitochondrial biogenesis, less is known about the ability of eNOS to regulate mitochondrial turnover. This turnover hinges on the intimately linked processes of biogenesis and autophagy – where dysfunctional, aberrant mitochondria are removed via selected autophagy (mitophagy) and replaced in an effort to maintain mitochondrial homeostasis within the cell . Indeed,in vitro siRNA-induced knockdown of eNOS in primary hepatocytes reduced fatty acid oxidation and impaired the induction of BNIP3 upon mitophagic stimulation. Collectively, these data demonstrate a novel role for hepatocellular eNOS in maintaining a healthy mitochondrial pool within the hepatocyte and may contribute to curbing NAFLD development (in vivo manipulation of hepatocellular eNOS is required to tease out its significance in hepatic mitochondrial dynamics and NAFLD development.The data on eNOS and autophagy/mitophagy are limited; however, there is strong evidence for NO regulation of autophagy. NO has consistently shown to be a potent activator of AMPK . This dielopment . FurtherOur lab has recently demonstrated that normal hepatic eNOS activation is dramatically reduced in the obese OLETF rat during the transition from hepatic steatosis to NASH , implicaOn the other hand, increasing NO bioavailability may provide some hepatoprotection against metabolic insults. Giving a systemic NO donor has recently been shown to attenuate NASH progression, in part through lowering M1 macrophage polarization  and promin vivo hepatocyte-specific gain and loss of function studies, and whether hepatocellular eNOS is required for hepatic mitochondrial adaptations to exercise. Uncovering a role for hepatocellular eNOS may improve our knowledge of NAFLD pathogenesis, but more importantly, also may provide a potential therapeutic target to attenuate hepatic mitochondrial dysfunction and curb NAFLD development.Significant strides have been made in recent years in teasing out the molecular mechanisms of NAFLD development and progression, including the pathological role of hepatic mitochondrial dysfunction. Given the well characterized role of eNOS in mitochondrial dynamics in an array of tissues, including exercise-induced mitochondrial adaptations, one could speculate that eNOS is also functioning to maintain a healthy mitochondrial pool in hepatocytes . Indeed,RC and RS drafted the manuscript. RC, RS, and RR edited and revised the manuscript. All authors contributed to the article and approved the submitted version.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.


In recent years, highly effective, well-tolerated, all oral direct-acting antiviral (DAA) therapies for hepatitis C virus (HCV) have raised the prospect of an era in which HCV elimination is achievable worldwide. Globally, 71 million individuals are estimated to be living with HCV. In 2016, the World Health Organization (WHO) issued viral hepatitis elimination targets that include diagnosing 90% of individuals living with HCV, initiating 80% of those who are eligible on treatment, and reducing incidence by 90%. While the WHO’s goals have been made more attainable due to the availability of these new effective therapies, recent modeling data suggest that 80% of high-income nations are not on track to achieve them by 2030 and nearly 70% are not expected to meet them before 2050 [Globally, HCV disproportionately affects individuals who intersect with the criminal justice system. Of the estimated 10.2 million people incarcerated worldwide on any given day, approximately 15%, or 1.5 million, are living with HCV [As the first steps in the care cascade, screening and confirmation of chronic HCV infection are important to ensure individuals are aware of their HCV status. Not only is HCV screening in correctional settings complicated by a lack of HCV-related knowledge, incarcerated individuals often do not provide a complete history of injection drug use due to the stigma associated with substance use disorders and fear of incrimination . TherefoFor individuals who are diagnosed, linkage to HCV treatment can be challenging. In general, correctional settings have even less access to DAAs than surrounding communities, particularly in low- and middle-income countries where global access is already poor. Even in nations with more widespread access to DAAs like the USA, the majority of facilities do not provide HCV treatment despite comparable outcomes in correctional settings , 7. MoreUpon release, justice-involved individuals face barriers related to social and structural determinants of health such as poor social support and housing, as well as relapse to active substance use. This makes it imperative to connect individuals to community-based social, behavioral, and addiction services prior to their release. One strategy is transitional care coordination, a patient navigation strategy that has proven successful to facilitate linkage to care for patients living with HIV . ImplemeBeyond justice-involved individuals themselves, the high rate of substance use disorders within this population indicates that this group plays a large role in community transmission. Therefore, HCV treatment in correctional settings not only benefits this high-risk population, but paves the way for preventing HCV transmission in the communities to which justice-involved individuals return. Correctional facilities can also provide HCV-related risk-reduction education to prevent new infections, which will not only lead to benefit for HCV, but should also be leveraged to prevent other blood borne and injection-related infections. Linkage to opioid agonist therapy and resources for syringe exchange will also be invaluable to these efforts.As HCV continues to disproportionately affect individuals involved with the criminal justice system, the development of effective strategies to promote testing, diagnosis, and linkage to care during incarceration is an opportunity to improve the coordination of care among this high-risk population. Strengthening the HCV-care cascade in correctional settings will move us closer achieving international reduction in new HCV infections, improving health outcomes for people living with HCV, and reducing HCV-related health disparities. To make HCV elimination conceivable in the coming decades, we must amplify international political will to tackle HCV in the criminal justice system; without action among justice-involved individuals, achieving the WHO elimination goals will be difficult to attain.


Unravelling spatio-temporal patterns of gene expression is crucial to understanding core biological principles from embryogenesis to disease. Here we review emerging technologies, providing automated, high-throughput, spatially resolved quantitative gene expression data. Novel techniques expand on current benchmark protocols, expediting their incorporation into ongoing research. These approaches digitally reconstruct patterns of embryonic expression in three dimensions, and have successfully identified novel domains of expression, cell types, and tissue features. Such technologies pave the way for unbiased and exhaustive recapitulation of gene expression levels in spatial and quantitative terms, promoting understanding of the molecular origin of developmental defects, and improving medical diagnostics. In this Review, Lisa Waylen and colleagues provide an overview of techniques used for spatial resolution of gene expression in a tissue or organ. They discuss the advantages, disadvantages and future directions of current methods and illustrate how spatial transcriptomics has impacted our understanding of biology. Spatial control of gene expression is crucial for defining tissue identity, from the patterning of a zebra’s stripes, to the left-right asymmetry present in most species, and deviations from this regulatory programme may result in developmental defects or disease, such as situs inversus or the variation in antigens expressed at different layers of a cancer tumour.3. Ectopic expression of Hoxa-1 in mice can reorganise regions of the hindbrain by transforming developing rhombomeres4, and in zebrafish, misexpression of Gata5 induces ectopic expression of myocardial genes such as nkx2.55. Of particular importance in development are morphogens, a concentration gradient dependent signal that provides a pattern for tissue differentiation and development. While some genes employ a simple on/off mechanism, many developmental genes respond to different expression thresholds to provide phenotypically distinct outcomes. In Drosophila, the maternal gene bicoid is responsible for regulating development of the anterior pole through graduated mRNA diffusion6 and limb patterning in the chick is established by morphogenic fields7.Our body plan relies on spatial expression, achieved by correct deployment of a developmental gene regulatory network (GRN) where the location, timing, and level of developmental gene expression are crucial. For instance, Hox genes are a highly conserved family involved in body axis patterning and provide an illustration of the requirement for specific timing, spatial restriction, and expression degree across species. Hox genes are silenced until activated by developmental timing cues, and sequential expression of Hox clusters will define the body plan of the embryo8, and subdomains of organs such as the kidney can be recapitulated from the preferential induction of progenitors9. This offers a unique platform to capture the spatial gene expression information required for accurate tissue patterning. Deviations from programmed, spatio-temporal gene expression can lead to disease, as seen in congenital disorders10 and highly heterogeneous cancer tumours which provide a prime target for spatial profiling analysis. For example, the dysregulation of HOX genes in leukaemia has been shown to support the immortalisation of malignant cells, while specific spatial expression patterns of biomarkers inform clinical prognosis and therapies12. In plants, changes in spatial gene expression have been shown to provide advance warning for the progression of viral infection13.Organoids raised from human stem cell cultures provide an invaluable model for exploring disease mechanisms spatially, by uncovering the cellular and molecular interactions occurring in three dimensions (3D). For instance, embryonic models such as gastruloids14 integrated bulk and computational methods with in situ hybridisation gene expression data, later accompanied by human and macaque atlases16. Despite challenging low resolution and lack of quantitative data, these atlases significantly advanced the current understanding of brain structure and function. More recently, the Allen Mouse Brain Common Coordinate Framework (CCFv3)17 sampled 1675 mouse brains at 10 μm voxel resolution, integrating multiple datasets in 3D. Virtual atlases are publicly available, browsable cell reference maps where gene expression data is reconstructed in 3D, digital space. They support myriad fields of research through interactive visualisation and analysis, providing a valuable source of information for many model organisms including C. elegans (Wormbase)18, frogs (Xenbase)19, Drosophila20 and zebrafish21.Therefore, to accurately decipher the GRNs involved in spatial gene expression, it is necessary to capture gene identity along with quantitative data. Previously, the Allen Mouse Brain Atlas constructed by the Allen Institute for Brain SciencesThis review explores current techniques used to elucidate spatial expression, from established robust methods with throughput limitations, to cutting-edge systematic and unbiased measurement of gene expression Fig. , Table 122. Large-scale in situ hybridisation is routinely used to simultaneously interrogate expression patterns of genes in sections or whole organs and tissues24. This is a particularly efficient method to screen for genes with regionalised expression patterns, such as those responsible for early growth determination25, but these techniques are low-throughput. At most a hundred genes can be simultaneously assessed, albeit in different samples. Another challenge of this traditional model is that visualisation of expression relies on probe binding efficiency, which varies significantly, eliminating the ability to perform comparative study between tissues or genes, without first performing background neutralising or equalising equations26. While in situ hybridisation addresses the need for spatially resolved data, accurate quantification of gene expression is often difficult to achieve using this approach and therefore requires further analysis.Historically, spatial patterns were captured from direct or in situ staining of genes of interest. In situ hybridisation (ISH) techniques Fig.  were dev27, which integrates the output from sequential single-molecule fluorescence in situ hybridisation and hybridisation chain reaction (HCR). HCR employs two complementary DNA probes which form a fluorescently labelled amplification polymer capable of relative quantification of mRNA expression28. In addition, this technology achieves higher spatial resolution due to the integrated single-cell imaging techniques. The SGA pipeline allowed novel characterisation of a pluripotent stem cell niche in development of the dorsal neural tube27. RNAscope provides a further integration of qualitative and quantitative approaches whereby probe hybridisation and signal amplification are performed in situ, allowing spatially relevant analysis at single-molecule resolution29. However, analysis is limited to a handful of target genes, since throughput from SGA is restricted by the number of distinguishable dyes.Further advancements to accurate quantitative measurement of in situ gene expression include the Spatial Genomic Analysis pipeline (SGA)(ClampFISH) was developed to improve the efficiency in FISH and the resulting weak signal response that demands high power microscopy. Indeed, the enzymes required in FISH may prove inefficient, providing inconsistent amplification or poor diffusion through the cell. ClampFISH detects unique nucleic acid molecules, and allows padlock-style probes to be used without enzyme support. Click chemistry is used to covalently circularise amplifier molecules by linking 5′ and 3′ ends30, topologically entangling individual amplifier molecules to increase binding specificity.Click-amplifying fluorescent ISH smFISH) relies on the use of many short probes which target multiple regions of the mRNA transcript. This can approximate the level of gene expression between different tissues, but is not numerically quantitative31. It is also limited by the need to bind sufficient primers to the query mRNA, which may not be possible for many short mRNAs. Although smFISH cannot assess non-coding RNAs such as those required for many regulatory functions, as it targets the ribosome/mRNA interactions, it can provide spatial localisation of RNA expression by directly imaging individual RNA molecules in single cells. Throughput is limited by the number of RNA species that can be simultaneously measured in single cells.Recent advances in ISH have introduced a novel approach to improve signal detection. Single-molecule fluorescence in situ hybridisation  relies on a non-barcoded approach focussed on designing image processing tools capable of processing large tissue areas and extensive data sets32. smFISH is more efficient at detecting RNA than single-cell RNA sequencing (scRNA-seq)33, and osmFISH has combined short hybridisation times and background reduction to attain high signal-to-noise ratios to further improve the sensitivity of RNA detection. osmFISH achieved a rate of 30% zero counts in contrast to the 82% registered by scRNA-seq, indicating its higher sensitivity to recover low levels of gene expression. This is largely because target number scales with the number of required hybridisation rounds to reduce error, and highly expressed genes do not affect the detection of non-highly expressed genes as barcoding is not required, and images can be analysed separately. FISH approaches rely on downstream image analysis to extract numerical measurements of gene expression. Meanwhile, scRNA-seq also requires reads filtering, mapping, and quality control to be converted into numerical measurements. osmFISH has been optimised for thin tissue sections, and a study of murine brain tissue developed an atlas of gene expression in the somatosensory cortex using 33 marker genes. Although a majority (59%) of the data came from neurons, many different cell types were observed and characterised.While previous studies in smFISH34 and assigns barcodes to individual RNA species, which can be imaged sequentially, allowing single-cell transcriptomic profiling. Branched DNA (bDNA) amplification substantially improved MERFISH detection efficiency, without increasing fluorescent spot size for individual transcripts, and it is now possible to use this method to study short RNAs35. This approach generates in situ transcriptomic analyses and detects >1000 RNA species, and provides error correction for >100. Further improvements to the efficiency of MERFISH gene processing allow simultaneous imaging of ~10,000 genes at a detection efficiency of ~80%36. While providing a high standard of subcellular resolution, the necessity for cell-dissociation challenges the spatial power of this approach.Multiplexed error-robust fluorescence in situ hybridisation (MERFISH) multiplexes 37. Reproducibility between sections is high, and result validation demonstrates a fair correlation with IHC, FCM and QIF, with some spatially restricted expression patterns of immune markers found to be associated with patient outcome38. A key advantage of this approach is that it is non-destructive, permitting reanalysis, and analysis of protein and RNA in cohort on serial sample sections. Digital Spatial Profiling is currently limited, with only four fluorescent visualisation stains available to examine tissue morphology and determine regions of interest.Digital Spatial Profiling Fig. , a new p39. A second reaction amplifies these tagged molecules, concatenates the copies, and adds new random nucleotides. While high signal intensity is a drawback to fluorescent imaging techniques, increased signal density is beneficial to DNA microscopy. Molecular proximities are computationally decoded from the overlapping diffusion fields generated by these signals, from which it is possible to infer physical images of the original transcripts at cellular resolution. Sequencing power permits detection of single-nucleotide variation, and spatially resolves biological features such as somatic mutation and stochastic RNA splicing. Variation in molecular density, however, makes it difficult to reconstruct images spanning large distances. Early applications indicate that DNA microscopy can function as an imaging medium equivalent to optical analysis, and is an important emerging approach to study the complex chemical dynamics of biological systems.Traditional spatial mapping relies on fluorescence light microscopy or direct physical registration of transcripts. DNA microscopy Fig.  instead,seqFISH+) serially reprobes a single slide to generate multiple images which can be computationally merged and analysed for a complete library of transcripts  allows a high-throughput accurate measurement of RNA transcript levels that can be achieved in an organ (bulk)42 or single cells through read-counts43. Data from bulk tissue analysis may mask the expression patterns of rare, or equally dominant cell types, or omit transient expression patterns. The quantitative data gained in RNA-seq is contrasted by the loss of spatial information. When isolated cells are spatially dislocated from their tissue of origin, transcripts lose spatial context; however, some studies have attempted to address these issues by using downstream cell sorting which reunites distinct cell populations. To achieve an analysis which benefits from data obtained from both spatial and quantitative methods, the two data sets need to be combined, a process which remains largely manual. Recent experiments to combine spatial information, with the quantitative data obtained from RNA-seq have been used to query the transcriptional changes in different regions of the cardiac infarct zone following heart injury in adults and neonates, providing type-specific quantitative expression data44.Quantitative methods of measuring accurate gene expression levels in high-throughput have been developed such as CAGE, SAGE, and gene expression arrays. CAGE sequencing permits analysis of coding and non-coding RNA expression by targeting promoter regions instead of translation interactions45. It relies on spatial segregation of landmark genes, identified through ISH experiments, to construct a reference map by including the RNA-seq data to quantify the expression level of these landmark genes. This map provides a transcriptome-wide reflection of spatial patterning which was used to identify key cells subpopulations in the Drosophila eye, including interommatidial cells and photoreceptors, each exhibiting a unique set of differentially expressed genes46.Mathematical models allow the integration of spatial and quantitative results by associating ‘landmark’ reference genes with transcript counts. For instance, Seurat is an algorithm that infers cellular localisation by integrating scRNA-seq data with in situ expression patterns47. A selection of brain cells from marine annelid P. dumerilii was profiled, and 81% of these cells were able to be mapped back to precise spatial locations. This approach relies on knowledge of ISH gene expression, and may not thoroughly encompass all embryonic domains. Both computational methods are powerful tools to generate data that is both spatially resolved, and quantitatively significant. However, these results are algorithm-based, rather than direct reflections of in situ measurements of the embryo.Moreover, one of the primary problems associated with scRNA-seq analysis, is the high technical noise of small-mass embryos, and subsequent low-copy number transcripts. To overcome this, the computations in Seurat also consider the spatial information of multiple genes that are co-regulated with landmark genes, thus providing a reliable gene expression map. Another computational method which integrates ISH with scRNA-seq provides a high-throughput approach to identify the tissue of origin for complex tissuesDrosophila melanogaster is, traditionally, a strong model for patterned expression with early emergence of a spatially defined embryo. Using a technique employing low-input RNA-seq, cryosectioned Drosophila embryos along the AP axis and isolated and sequenced mRNA, spatial patterns were observed to closely match previously determined in situ and microscopy patterns48. This method was used to generate a time course of the developing embryo. Many genes not previously characterised as spatial genes were found to exhibit spatially restricted expression, such as pole specific genes with no functional annotation. This approach experienced a large degree of noise, as too much carrier RNA gave only a small number of reads per slice. Expression was measured across spatially resolved tissue slices, leading to the blending of cell signals and providing poor resolution of cell populations that did not reach the single-cell level. While spatial landmarks could be identified, spatially intermixed populations could not be well defined. Computational approaches can be employed to spatially reconstruct complex tissues without needing prior annotation, a process used to investigate maternal factors with mutants49, however, this still provided lower resolution results than in situ analysis, and it was difficult to observe transitional domains.DistMap  is effective in rejecting errors. Using this approach, a 160-gene set was detected and quantified in mouse primary visual cortex, where clustering revealed distinct cell types, overlaid with spatial expression distribution across layers of cortex. Upregulation of activity-regulated genes in response to visual stimulation was observed. Gene expression results were found to correlate well to in situ hybridisation and scRNA-seq. This technique is scalable to larger 3D tissue blocks and can be adapted for higher gene numbers, with current best throughput at ~1000 genes. The challenge to sequence all genes simultaneously remains.A key benefit of scRNA-seq is the power to detect biological variability between cells, and define characteristics of rare cell types. While in situ RNA-seq is able to provide spatial context, throughput efficiency is limited and is difficult to scale to whole tissues. The map Fig.  approachTomo-seq , followed by bulk RNA-seq of each section. Triangulation algorithm, RNA-tomography, is then automatically applied to estimate gene expression levels at the intersection of the three sections, creating a digital three-dimensional expression pattern of the whole embryo. Depending on section thickness, this mathematical reconstruction could recapitulate gene expression at a resolution equivalent to single-cell analysis. However, the accuracy of this computed gene expression value needs to be validated with scRNA-seq datasets. Tomo-seq necessitates using identical samples for an accurate overlay of sections on multiple axes. As this is biologically difficult to accomplish, there is room for a degree of error in combining tissue sections. Applied to developing zebrafish embryos at three developmental stages , Tomo-seq delivers a comprehensive digital resource for embryonic gene expression in the whole embryo. Approximately 20% of the zebrafish transcriptome exhibited spatially restricted gene expression, a large number of which were functionally uncharacterised52. Combined with chromosome conformation capture datasets, Tomo-seq was used to identify spatially co-expressed genes correlating with topological domains53. Pre-zygotic transcription exhibited strict structuring with super enhancers clustering during development, a pattern which is similar to that previously observed in mammals. Super enhancer regions present a greater degree of transcriptional regulation, and are associated with more highly expressed genes54. Tomo-seq has also been successfully applied to an entire isolated organ. Indeed, a high spatial resolution map of the embryonic zebrafish heart obtained with Tomo-seq revealed 1100 genes being differentially expressed in the various sub-compartments of the developing heart, 502 of which were previously uncharacterised. Further examination of the sinoatrial pacemaker region of the heart revealed a novel role for Wnt/β-catenin signalling in the pathway regulating autonomic control, which provided useful insights into the molecular basis of heart function. This illustrates the discovery potential of this technique to search for novel gene targets for therapeutic use55.seq Fig.  automatiGeo-seq  and not possible with NGS-based methods. By processing different tissue domains within the same reaction, Spatial transcriptomics is able to remove much of the technical variation experienced by many standard expression methods. Application of this technique in murine brain tissue demonstrated gene clustering that correlated with morphological layers, allowing identification of cluster (and subsequently tissue) specific markers, while analysis of human breast cancer cells revealed that invasive cells exhibited high expression of extracellular-associated genes, and an unexpectedly high degree of cellular heterogeneity within biopsy samples, information important for decisions about prognosis59. The integration of Spatial transcriptomics with scRNA-seq from dissociated whole human embryonic hearts was able to identify a novel population of Myoz2-enriched cardiomyocytes in both the atria and ventricles which complemented recent findings in mice, yet to be observed in humans60. Currently, the resolved power of Spatial transcriptomics remains limited by the spacing of primers required for barcoding.ics Fig.  is the lSlide-seq  which employs 2 μm spatial barcoding63 and permits histological analysis which is not possible with Slide-seq.seq Fig.  alleviatSlide-seq data using non-negative matrix factorisation regression (NMFreg) which reconstructs Slide-seq expression as a combination of cell-type signatures from scRNA-seq61. LIGER  is a computational method for spatially locating cells present in scRNA-seq data from in situ transcriptomic data, thereby increasing the resolution of the in situ data64. The Harmony algorithm, which projects cells into a shared embedding in which cells group by cell type rather than dataset-specific conditions, is shown to be both efficient and accurate65. Another multiple datasets integration pipeline, inspired by multiple sequence alignment, utilises canonical correlation analysis (CCA) to identify anchor points across heterogeneous datasets66. An alternative approach is SpaOTsc, which utilises genes with spatial measurements to extrapolate the spatial properties of scRNA-seq data67. A different approach is found in Giotto, a user-friendly workspace that utilises cell-type-specific gene signatures to infer cell-type enrichment scores for downstream analyses with the capability for integration of spatial information68. Altogether, these computational approaches present timely developments to capitalise on the rapid growth of high-throughput tissue sectioning-based technologies and are summarised in Table It is possible to map scRNA-seq data onto novoSpaRc is a recent gene expression cartography technique that performs mapping based on the variation of gene expression across a tissue section, using a probabilistic optimisation technique69. However, the accuracy can be sub-par due to the lack of reference map, and a set of a priori marker genes with known expression patterns is desirable. In Drosophila, ScoMAP  is another reference-free technique that spatially integrates expression data into a virtual latent space, resembling the organization of a 2D tissue70. At the single-cell resolution level, the CSOmap  algorithm can partially reconstruct the tissue spatial organisation based on ligand-receptor interaction71. Altogether, these computational advances demonstrate the potential power of in silico techniques in de novo spatial mapping of expression data, but there is still room for improvement in prediction accuracy.Rather than combining two different datasets, a new class of advanced computational techniques enables the prediction of spatial information from a single gene expression dataset. Recently, the emergence of high-throughput single-cell resolution 3D analysis methods has provided the spatio-temporal expression information necessary to understand embryogenesis and development. The main advantage of these techniques over the previous integrating methods, is that these do not require prior annotation, nor do they rely on tissue dissociation to obtain quantitative values.novoSpaRc sly Fig. , by sequ73. Progressing beyond curated databases, 3D-visualisation cell atlases modelling detailed spatial data will provide the most benefit in research and medical applications74. Several applications exist which allow high fidelity imaging datasets to be integrated with quantitative data for completely interactive analysis, such as Virtual Fly Brain75, and the atlas of embryonic human hearts which combines Spatial Transcriptomics and scRNA-seq60. Results from DistMap were used to generate Drosophila Virtual Expression eXplorer (DVEX), an online, browsable database which generates virtual ISHs capable of predicting spatially resolved expression patterns in stage 6 embryos. Expression data is currently limited to binarized bins under defined thresholds, each expressing 6500–8500 genes, and cells are mapped independently with no incorporation of previously mapped cells into score refinement. Other 3D-visualisation tools such as MorphoNet76 will assist in integrating quantitative information from different platforms or sources on the same 3D object.There is high demand for public repositories of spatially resolved transcriptomic data, which allow comparison and integration of multiple sources and resolutions of analysed output77. Fortunately, the majority of analysis tools in spatial transcriptomics are publicly available on GitHub , paving the way for building community standards, potentially enabling integration of different spatial transcriptomics technologies to address a common biological question. Further, it has become standard practice in the research community to have preprints publicly available , allowing rapid dissemination of methods and results, and lessons can be learned from the systems biology standards79. This presents a great opportunity and a critical goal for the spatial transcriptomics community now that various technologies are in the developmental phase.To promote reproducibility, standards for data storage, analysis and exchange are urgently needed, as spatial transcriptomics currently lack common guidelines for data management, compared to more mature sequencing technologies 80. Tools have continued to develop, with various improvements to the efficiency, versatility and power of these non-invasive imaging approaches83. Recent improvements in single-molecule detection rendered this technology amenable for quantitative measurements and larger throughput, forecasting future scalability to assess whole transcriptomes.Standard techniques such as in situ hybridisation are widely used to interrogate spatial gene expression. Based on visual examination, these techniques are extremely powerful in detecting changes in gene expression patterns. While providing useful data spatially, the quantitative power of fluorescence imaging remains low84, employing “multimodal intersection analysis” to capitalise on the strength of information about cellular identity gained from scRNA-seq and the spatial data from microarrays to highlight spatially restricted gene networks and cell enrichment. Such approaches pave the way towards establishing virtual atlases which achieve a whole-organism transcriptome with cellular resolution. Several single-cell atlasing projects are endeavouring to develop these public resources which provide a valuable source of information for many model organisms including The Human Cell Atlas85 and single-cell zebrafish transcriptome atlas86. Ultimately, spatially resolved transcriptomics will be paramount in adding spatial coordinates to these single-cell atlasing projects87.RNA sequencing and its derivative technologies have answered the need for quantitative data and are suitably high-throughput for assaying whole genomes. These methods, however, lack the acuity necessary to deliver spatial results. Combining ISH and scRNA-seq using computational integration, achieves the level of accuracy required for resolving accurate spatio-temporal gene expression pattern. Identification of spatial markers and archetypal expression patterns is the basis for these current integrative approaches. Cell types can be visualised in 3D with computational techniques, however, this is based on measurement of spatial information from 2D slides and careful sampling is required to ensure faithful recapitulation of spatial patterning. Challenges remain in scaling these methods to accommodate large tissue volumes, however, as sequencing costs drop, it is forecast that it will be feasible for these technologies to scale to whole organisms. High-resolution, imaging-based approaches require high magnification and fine sectioning necessitating long imaging times, which will protract further with scaling. Methods which employ targeted probes carry the inherent limitation of finite available fluorophores, and will be challenging to scale. Underpinning these challenges is that, as more cells and tissues are analysed with higher resolution, more data points are required, demanding higher computational power, and scalable mathematical models for future 3D visualisation and data interpretation. Spatial technologies vary in their readiness for scaled analysis, and this will prove a key determinant of the lifespan and relevancy of the technology. Further limitations remain such as the difficulties in obtaining reproducible results due to single-cell analysis variability, and the applications in heterogeneous tissues such as eye retinas and cancer tumours remain to be determined. Methods to integrate spatial measurements with scRNA-seq data are beginning to emerge88. Ongoing modifications and novel data integration enhance utility, providing new versions of existing technologies such as Slide-seqV2 which is currently in development89.Accessibility to spatially resolved methods will determine popularity and speed of incorporation into research. Commercialisation of novel technologies supports consistent and reliable results, and promotes competition-driven enhancements to sensitivity and efficiency. CARTANA (Sweden) offers kits and servicing for padlock-based in situ sequencing technologies, while 10X Genomics acquired Spatial Transcriptomics and their Visium Spatial Gene Expression system reduced barcode spacing, leading to an improved resolution and 55 µm microarray90. To generate a transcriptomic map capable of capturing the complexity of gene expression, it must be possible to detect a range of RNA molecules, unrestricted by length, and have the ability to distinguish between similar isoforms. Technologies which lack sequencing capabilities, also lack the ability to discriminate between many alternate and splice forms, and cannot faithfully predict the effect of RNA structure. Approaches like smFISH which require multi-probe binding, restrict the discovery of short sequences of RNA such as microRNA and tRNA, and limit the profiling of different isoforms. Technologies which fail to capture subtle variations in sequence length and identity, or which lack the resolution to distinguish unique isoforms of RNA, comprise the complexity of these systems, and novel insights regarding cell type and gene function may be overlooked.Several types of RNA molecules are active within the cell, engaging in diverse roles from mRNA encoding proteins, to non-coding and small RNAs regulating gene expression at the transcriptional, post-transcriptional and epigenomic levels91. Computationally aligning spatially registered images with quantitative expression data will allow a unique appreciation of the complexity of tissue structure and composition. High-throughput spatial transcriptomics are becoming an essential component of both bench and bedside medicine, with an increasing demand for efficient analysis pipelines in clinical histology and pathology. The ultimate goal is to generate spatial-omics data, where a single sample may be non-destructively analysed to reveal temporal, spatial, and quantitative data simultaneously.Rather than supplant existing techniques, many new approaches are designed to integrate with current technologies, expanding the scope of retrieved data. Many fields of science provide valuable insights into spatial genomics, however, without a wide-ranging, collaborative effort, these assets will remain underutilised. Mass spectrometry imaging is one such avenue yet to be fully explored for its integrative potential, despite demonstrating clear benefits in applications such as proteogenomics where structural imaging is followed by RNA sequencing92. Recent reports capturing expression data in vivo by tracking transcription factor binding have illustrated the possibilities of these techniques93, including developing predictive GRNs94. Integrating 3D regulatory information, such as epigenomics studies, will complete our appreciation of the GRN controlling spatio-temporal gene expression patterns. Advances in epigenomic studies to allow single-cell analysis, such single-cell ATAC-seq95 have provided new insights into regulatory heterogeneity in complex tissues96. Ultimately, incorporating chromatin architecture and regulation in a 3D digital embryo will provide a comprehensive framework to uncover the GRNs executing spatio-temporal control of gene expression97. This promises to accurately predict the downstream effects of genetic perturbations, such as those caused by mutations resulting in developmental disorders or disease, in an immediate, automated manner.Integration of temporal expression datasets will enhance understanding of spatial transcriptomics. Incorrect temporal expression patterns can lead to developmental disorders or diseases like cancer. Understanding the gene expression timescale is crucial to identifying key triggers at different stages of development. For instance, single-cell studies performed at different stages of pluripotent cells differentiating into cardiomyocytes, revealed novel developmental pathways underlying lineage specification during cardiogenesisPeer Review File