Postmastectomy radiotherapy is considered to be a necessary treatment in the therapy of breast cancer, while it will cause soft tissue damage and complications, which are closely related to the success rate and effectiveness of breast reconstruction. After radiotherapy, cutaneous tissue becomes thin and brittle, and its compliance decreases. Component fat grafting and adipose-derived stem cell therapy are considered to have great potential in treating radiation damage and improving skin compliance after radiotherapy.In this paper, the basic types and pathological mechanisms of skin and soft tissue damage to breast skin caused by radiation therapy are described. The 2015–2021 studies related to stem cell therapy in PubMed were also reviewed. Studies suggest that adipose-derived stem cells exert their biological effects mainly through cargoes carried in extracellular vesicles and soluble secreted factors. Compared to traditional fat graft breast reconstruction, ADSC therapy amplifies the effects of stem cells in it. In order to obtain a more purposeful therapeutic effect, proper stem cell pretreatment may achieve more ideal and safe results.Recent research works about ADSCs and other MSCs mainly focus on curative effects in the acute phase of radiation injury, and there is little research about treatment of chronic phase complications. The efficacy of stem cell therapy on alleviating skin fibrosis and its underlying mechanism require further research. Radiotherapy plays an important role in the treatment of malignant tumors. Postmastectomy radiotherapy (PMRT) is a more commonly performed and safer option. An early comprehensive meta-analysis  analyzedHowever, radiotherapy inevitably causes soft tissue damage and complications, which are closely related to the success rate and effectiveness of and satisfaction with breast reconstruction –4. AfterThe decision of whether to perform radiotherapy as well as the sequence of radiotherapy and breast reconstruction is closely related to the recurrence of breast cancer and the quality of breast reconstruction. The sequelae of radiotherapy hinder reconstruction, and clinicians must therefore make a trade-off between safety and reconstruction quality. Therefore, research of how to prevent skin and soft tissue damage hindering breast reconstruction after radiotherapy is of great value.In recent years, autologous fat grafting has attracted much attention based on its small residual scar and convenience. There are gradually some studies indicated that autologous fat grafting can partially improve the problem of poor skin compliance after radiation fibrosis –11. ADSCRadioresistance varies according to the stage of the cell cycle. Cyclin-dependent kinase inhibitors, such as P16, P21, and P53, are common checkpoint proteins expressed during the radiation-resistant phase.In G1 phase, these inhibitors are expressed to ensure that the cell successfully enters the DNA synthesis phase (S phase). Once the cell enters S phase, these inhibitors and enzymes responsible for DNA repair are overexpressed to ensure the accuracy and integrity of DNA synthesis. Due to residual levels of S phase enzymes and cyclin-dependent kinase inhibitors, cells in G2 phase are also considered to be radiation-resistant.In early M stage, chromosomes condense, and DNA is very susceptible to radiation damage. In general, there is a lack of repair mechanisms in M phase, and condensed and concentrated DNA is a perfect target for ionizing radiation. Therefore, M phase cells, i.e., dividing cells, are particularly radiosensitive .Owing to this characteristic, cancer cells are the main target of radiotherapy. However, in addition to cancer cells, some normal cells that divide vigorously, such as stem cells, basal skin cells, and gastrointestinal mucosa cells, are also targeted by ionizing radiation. Unrepaired DNA damage often induces apoptosis and cell cycle arrest , 15. CelReactive oxygen species (ROS) are an important cause of indirect damage induced by radiation. Ionizing radiation causes the transfer of electrons when it penetrates cells, leading to generation of unstable ROS . In addiExtensive research has found that radiation-induced damage arises at sites outside the irradiated area . This inIn addition, the radiation-induced inflammatory response is also considered to be important for indirect injury. The acute inflammatory response can cause secondary tissue damage, and the chronic inflammatory response is closely related to radiation-induced soft tissue fibrosis .In the short term, cell damage caused by ionizing radiation induces an acute inflammatory response, and the associated inflammatory cells are mainly neutrophils and macrophages . AdipocyAccording to Koenig et al. , in addiUltrastructural analysis showed that the basal membrane of capillary vessels duplicates and their lumens are ectatic after radiotherapy. Endothelial cells are rich in cytoplasm, including macropinocytotic vesicles and a large number of Weibel-Palade bodies. Many adipocytes are necrotic. A large amount of accumulated collagen and debris from necrotic adipocytes in connective tissue is visible .The acute phase response is closely related to endothelial cell damage caused by ionizing radiation Fig. . IonizinNeutrophils play an important role in endothelial injury during acute inflammatory response. The first step in causing damage is to enhance the interaction between neutrophils and endothelial cells. Normal neutrophils have low expression of α4-integrin. Neutrophils under the action of multiple inflammatory factors up-regulate the expression of α4-integrin and enhance the interaction with endothelial cells by acting on VCAM-1 on the surface of endothelial cells. In addition, cytokines such as TNF-α and IL-1 also up-regulate the expression of adhesion molecules such as vascular cell adhesion molecular 1(VCAM-1) and intercellular adhesion molecular 1 (ICAM-1) –39. Afte2+. The increase in intracellular Ca2+ activates a disintegrin and metalloprotease 10 (ADAM10), finally causing the degradation of vascular endothelial-cadherin (VE-cadherin) and increasing the endothelial permeability [Endothelial cells stimulated by ionizing radiation and inflammatory signals also increase permeability. ROS produced by ionizing radiation raise the concentrations of intracellular Caeability –47. It cAs the most commonly reported complication of breast carcinoma management, the incidence and severity of lymphedema are closely related to axillary radiotherapy and axillary lymph node dissection. However, for most invasive breast cancers, axillary treatment remains indispensable for safety reasons . Three tThere is much speculation and research about the mechanism underlying radiation-induced lymphedema. However, contrary to initial assumptions, early studies performed in vitro and in vivo in humans and other mammals demonstrated that lymphatic vessels are relatively insensitive to radiation . By contRadiation-induced fibrosis results in progressive functional and cosmetic impairment, which is a common late complication of PMRT Fig. . RadiatiDue to the body's repair mechanism, vascular endothelial cells damaged by radiation exhibit high levels of intracellular trafficking and transport between themselves and their environment and high permeability, and vascular basement membrane replication and lumen expansion are also observed. Therefore, the acute phase of injury is mainly manifested as an inflammatory reaction of blood vessels, such as erythema and edema . This leIn addition, radiotherapy can cause an abnormal morphology of fibroblasts, such as a flattened and enlarged morphology, as well as changes in their biological behavior and synthetic function, leading to modulation of important signaling pathways and adaptive dysfunctions. Fibroblasts injured by radiotherapy exhibit inhibited proliferation but increased migration, invasiveness, adhesion, and contractility. At the same time, activation of the HIF-1α pathway also initiates a local fibrotic response . A studyDecreased skin compliance due to radiation fibrosis may hinder breast reconstruction, for example, may reduce the retention of grafted fat and increase the probability of capsular contracture in breast implant reconstruction . ElevateIn the process of repairing and rebuilding, it is necessary to solve the problem of lack of breast volume and the problem of cell dysfunction and tissue damage caused by radiotherapy. Autologous fat grafting has attracted much attention for these two advantages. It can obviously improve skin tissue suffered from irradiation by improving the compliance and thickness of skin and tissue adhesion , 54, 63.ADSCs, a type of mesenchymal stem cell (MSC), have the potential of multi-directional differentiation and can differentiate into adipocytes, chondrocytes, osteocytes and endothelial cells. Under specific conditions, ADSCs can replace damaged cells through proliferation and differentiation to repair tissue damage , 68.The mechanisms regulating the proliferation and differentiation of ADSCs are not fully understood. Lin et al.  indicateADSCs can secrete various cytokines, chemokines, growth factors, and paracrine molecules in extracellular vesicles and promote cell survival, modulate the inflammatory reaction, and thus enhance regeneration of injured tissue , 72.Many studies have explored the role of MSCs, represented by ADSCs, in repair of radiation injury and other soft tissue injuries Table .Table 1TRAF6 and IRAK through the NF-κB pathway, while miRNA-34 [IL-6 and TNF-α by targeting the Notch1 pathway. The anti-oxidative stress [ADSCs can increase proliferation and survival of specific stem or progenitor cells in tissues and organs  and prommiRNA-34  inhibitse stress  and antie stress  effects e stress , 96, Nrfe stress –99 and sStudies suggest that ADSCs exert their biological effects mainly through cargoes carried in extracellular vesicles (exosomes or microparticles) and soluble secreted factors . There aFinally, most current research about ADSCs and other MSCs mainly focuses on curative effects in the acute phase of radiation injury, and there is little research about treatment of chronic phase complications. For breast reconstruction, most patients are in the chronic phase of radiation damage, and fibrosis is relatively severe. The efficacy of stem cell therapy to improve skin fibrosis and skin compliance and the underlying mechanism require further research.ADSCs can promote wound healing and tissue repair. However, the mechanism by which ADSCs improve radiation injury and their ideal application method in this context must be studied. Further research is also needed to determine whether ADSC therapy is efficacious and safe for breast reconstruction after radiotherapy and, if so, to examine the underlying mechanism.


Cancer seriously threatens human health. Surgery, radiotherapy and chemotherapy are the three pillars of traditional cancer treatment, with targeted therapy and immunotherapy emerging over recent decades. Standard drug regimens are mostly executed via intravenous injection (IV), especially for chemotherapy agents. However, these treatments pose severe risks, including off-target toxic side effects, low drug accumulation and penetration at the tumor site, repeated administration, etc., leading to inadequate treatment and failure to meet patients’ needs. Arising from these challenges, a local regional anticancer strategy has been proposed to enhance therapeutic efficacy and concomitantly reduce systemic toxicity. With the advances in biomaterials and our understanding of the tumor microenvironment, in situ stimulus-responsive hydrogels, also called smart hydrogels, have been extensively investigated for local anticancer therapy due to their injectability, compatibility and responsiveness to various stimuli . Herein, we focus on the latest progress regarding various stimuli that cause phase transition and drug release from smart hydrogels in local regional anticancer therapy. Additionally, the challenges and future trends of the reviewed in situ smart hydrogels for local drug delivery are summarized and proposed.  Cancer is a global health problem and has become a leading cause of mortality ,2. CurreLocal administration of anticancer drugs is characterized by lower drug dose requirements and less toxicity to non-targeted tissues compared with systemic administration, which is expected to achieve greater efficacy and fewer systemic side effects. Various formulations have been investigated to directly distribute drugs to the tumor site, such as gels, suspensions, particles and films ,7,8,9. AIn the context of local administration, the injectability of hydrogel facilitates localized application, avoiding surgical injury. Injectability can be achieved by in situ sol–gel phase transition of the hydrogel composition once it is injected into the tumor site, thus forming drug depots for sustained drug release ,12. SmarThe present review aimed to explore the most recent advances in situ smart hydrogels for local antitumor therapy . First, Intravenous drug delivery remains the dominant route of administration for cancer treatment. However, the greatest challenges of intravenous drug delivery, namely poor targeting that induces low tumor accumulation and high systemic toxicities to healthy organs, continue to confound oncologists . In addiOver the last two decades, ongoing efforts have been made to implement targeted therapies, such as nanotechnology, which have been designed to improve the efficacy and reduce side effects of chemotherapy through passive or active targeting ,16. HoweLocal delivery of chemotherapeutic agents can enrich the accumulation of drugs in the injection site and reduce systemic toxicity, allowing low doses to achieve low toxicity and high efficacy . NeverthHydrogels can be classified by various parameters, such as the shape and size, the charge of the hydrogel-forming macromolecules and the mechanism of crosslinking during hydrogel formation ,26. The Temperature-responsive hydrogels, which are capable of responding to temperature changes, have been extensively studied among the smart hydrogels ,32. As tFor LSCT hydrogels, the polymer matrices are miscible with water at a temperature lower than the LCST, but the solubility drops dramatically at or beyond the LCST, and then a sol–gel phase transition occurs a 10]. H. H10]. HIn contrast to LCST systems, the phase transfer in UCST systems is reversed. Briefly, as the temperature rises, the solubility of the polymer matrix increases, and the system exhibits a gel-sol transition at the USCT. Notably, the phase transition temperature of most USCT systems is usually below 25 °C . HydrogeThermoresponsive hydrogels are based on natural  or synthetic  macromolecules. We consulted the most recent literature on studies of temperature-responsive hydrogels for intratumoral drug delivery in tumor treatment. The polymer materials, therapeutic agents, as well as in vitro and in vivo cancer models of some of the latest reported thermoresponsive hydrogels are summarized in Chitosan (CS), a cationic polysaccharide, produces thermoresponsive hydrogels through interactions with anionic molecules such as β-glycerol phosphate disodium salt (β-GP) at physiological temperature . For insPoloxamers, triblock copolymers of PEO-b-PPO-b-PEO, are classic thermosensitive and biodegradable synthetic polymers. The LCST of poloxamers is close to physiological temperature when the molecular weight, composition and concentration are adequate, which is important in biomedical applications. Micelle formation theory could be used to explain the gelation mechanism due to the amphiphilicity of poloxamers ,39. TherAlthough poloxamers are biodegradable and biologically inert, the development of hydrogels made from poloxamers has been limited by the low viscosity and poor mechanical properties. Accordingly, significant efforts have been made to construct thermoresponsive hydrogels based on other synthetic polymers, such as PCLA-PEG-PCLA , PLEL 11 and PEGThe acidic tumor microenvironment is one of the main characteristics of almost all solid tumors due to the enhanced production of lactate by tumor cells resulting from their high rate of aerobic glycolysis . Such ac+) by accepting protons because of the acidic external pH of the tumor microenvironment. The chains of the polymer will expand and swell as a result of the electrostatic repulsion between charges  by Zhang et al. developed an injectable pH-responsive drug–peptide hydrogel for local chemotherapy . FirstlyThe formation of polyelectrolyte complexes between anionic and cationic polymers is another method of preparing pH-sensitive hydrogels. Many natural polymers  and synthetic polymers  have been studied to produce pH-sensitive hydrogels ,54,55. AIn addition, some dynamic covalent bonds are pH-responsive groups as well. For instance, polymers abundant in hydrazone bonds are stable at a normal physiological pH (pH 7.4) or under alkaline conditions, whereas the chains will rapidly cleave at a mildly acidic pH. Thus, polymers with such hydrazone bounds will undergo a phase transition as the pH changes. Jia et al. designed a hydrogen bond and hydrazone bond crosslinked pH-responsive hyaluronic acid–collagen (HA–COL) hydrogel, which achieved a sol–gel phase transition quickly in neutral and alkaline environments . In vitrIn recent years, peptides, especially self-assembled peptides, have emerged as important structural elements of the hydrogel networks because of their remarkable properties such as versatile functionality, biocompatibility and biodegradability ,67,68. ILight-responsive hydrogels undergo property changes and a phase transition as a consequence of light exposure, including near-infrared radiation (NIR), visible light and UV . AccordiYang et al. prepared sodium selenite (Se)-directed crosslinked hydrogels based on HA–dopamine loaded with the photothermal agent indocyanine green (ICG) for localized breast cancer therapy . After N3+ ions with UV irradiation ,9594,95]Enzymes are substances with specific catalytic activity and play an important role in almost all metabolic processes in the human lifecycle. The type, activity and quantity of enzymes are different in the physiological or pathophysiological conditions of different tissues and organs. Enzymes, as signals to modulate metabolism, nutrition and energy conversion, are essential during tumorigenesis and tumor development. Importantly, the level of several enzymes in tumors significantly differs from that in normal tissues . Thus, d2O2 and horseradish peroxidase (HRP) [Matrix metalloproteinases (MMP) have been found to be overexpressed in a variety of tumors, and are among the factors most widely used to activate smart drug carriers . Li et ase (HRP) . An in vElectricity-sensitive hydrogels can undergo property changes including swelling, deswelling, bending or erosion in response to electric field exposure . Such elQu et al. designed a biocompatible biodegradable conductive hydrogel with the ability of precise “on–off” drug release in response external electrical stimuli . An elecA low current can induce charged substances in the hydrogel composed of polyelectrolytes to move, inducing a change in the volume of the hydrogel and drug release, which is especially suited for drug delivery across the skin in melanoma therapy. Interestingly, some polyelectrolytes can self-polymerize to form in situ hydrogels by electrostatic interactions in the absence of electric fields. Park et al. proposed a novel strategy based on electrostatic interactions using cationic chitosan and anionic HA to prepare a Substance P (SP) analog (SP1)-loaded injectable hydrogel system . The sepApart from the abovementioned smart hydrogels, other stimulus-responsive in situ hydrogels have been investigated for local regional cancer treatment as well. The in situ hydrogel matrix undergoes physical or chemical changes in response to stimuli such as glutathione , and ant3O4 nanoparticle (Fe3O4 NP)-grafted graphene oxide (GO) [In addition, dual and multiple responsive hydrogels for localized antitumor therapy have received more attention than single responsive hydrogels in recent years with the increasing demand for precision therapy. Given the complex tumor microenvironment, these hydrogels are designed to respond to two or more of the abovementioned stimuli for more precise control over the responsive behavior and improvement in the overall performance and applicability of localized drug delivery. Yuan et al. constructed a hydrogel (PNAGA-DMP-Fe3O4@GO) with multiple responsiveness to NIR, light and/or enzymes with thermosensitive poly(N-acryloyl glycinamide) (PNAGA) loaded with doxorubicin (DOX) and polyester (PE) capped mesoporous silica nanocarriers (MSNs) as well as Feide (GO) . This muide (GO) . The resDespite these reported multi-stimulus–responsive in situ hydrogels exhibiting excellent antitumor effects and negligible side effects, how to achieve individualized and precise treatment and ensure the reproducibility of the therapeutic effects remain a hard nut to crack. Multi-stimulus–responsive hydrogels are often connected with complicated design and fabrication procedures, which are very common obstacles for novel drug delivery systems being applied in the clinic. Accordingly, how to balance the complex design of in situ smart hydrogels with simple scaled-up industrial production is a very challenging direction for the future.The present review mainly provides an overview of injectable smart hydrogels responding to various stimuli, including temperature, pH, light, magnetic fields, enzymes, electric field, etc. These hydrogel composites change their properties, and exhibit phase transition and controlled drug release because of the special physical and/or chemical properties of the polymer matrix. Local-regional therapy via in situ smart hydrogel systems has been demonstrated by a large number of studies to improve the therapeutical outcomes with fewer adverse effects. Additionally, hydrogels with dual or multiply responsiveness, usually constructed as a combination therapy strategy, have also been proven to further enhance treatment outcomes and weaken toxicity. Hence, in situ smart hydrogels for local administration have been considered as a novel and promising strategy for anticancer in recent decades.Most localized therapy strategies of in situ hydrogels are mainly limited to superficial tumors and are unavailable for distant lesions or metastases or deep tumors because external stimuli such as light are unable to penetrate deep tissues. The intratumoral injection technique for deep tumors is also a problem for local administration. Optimistically, with the aid of modern imaging techniques such as ultrasound, CT guidance or laparoscopy, safe and precise injections can be achieved in some deep solid tumors.Smart hydrogels have been advanced in recent decades with the development of biomaterials. Although the short-term biocompatibility and safety of most new synthesized biomedical materials have been assessed, the long-term toxicity remains inconclusive and requires further investigation and clarification. Therefore, it can be predicted that the safety, especially the long-term safety of biomedical materials, will become an essential research aspect in the future.The responsiveness of hydrogels to the stimuli in human tumor tissues might be different from that observed in the laboratory. Even in vitro experiments under pathophysiological conditions sometimes might not be the same as in vivo studies. Moreover, the in vivo reproducibility of the response of smart hydrogels can be influenced by individuals at different cancer stages. Thus, designing personalized and precisely tunable hydrogel systems with dual-stimulus or muti-stimulus responses to meet different patients’ needs is an important and promising direction. Notably, smart hydrogels that are sensitive to biological factors such as enzymes or antibodies have great potential for conduct precise and individual anticancer therapy in combination with other stimuli, which allows precise targeted delivery of the loaded drug.The clinical translation of such in situ hydrogel systems still remains difficult. Numerous studies have stopped at the stage of preclinical studies, while a few in situ smart hydrogels have entered clinical trials. This might be ascribed to the complex synthetic routes of some polymers and the complicated preparation process of drug-loaded hydrogels, resulting in difficulty in industrial-scale production. Accordingly, the design of simple and single-step preparations of such hydrogels is another promising direction. Moreover, standardized operation and testing processes are indispensable for realizing industrial production and ensuring their reproducibility.Although the recent decades have witnessed hundreds of successful examples of injectable hydrogel applications for local-regional therapy, challenges in the development of these hydrogel platforms are still present. Several obstacles that need to be addressed soon and potential development prospects are listed below:Generally, the design and development of injectable in situ smart hydrogels have received increasing attention and represent an attractive research direction in the field of local regional cancer therapy. In contrast to traditional or commonly used therapeutic strategies, smart hydrogel systems exhibit specific characteristics: injectability, in situ phase transition and controlled drug release in response to various stimuli. If we take all these together, in the foreseeable future, it is reasonable to believe that a growing number of these hydrogels will enter clinical trials and application for cancer therapy in the future.


Attention deficit hyperactivity disorder (ADHD) is a prevalent neurodevelopmental disorder that can diminish the quality of life of both children and adults in academic, occupational, and social contexts. The kynurenine pathway (KP) contains a set of enzymatic reactions involved in tryptophan (TRP) degradation. It is known to be associated with the risk of developing ADHD. This review will address the KP and underlying mechanism of inflammation in ADHD. Potential inflammatory biomarkers reported in the most recent studies are summarized. Although a strong neuroimmunological basis has been established due to the advances of recent neurobiological research, the pathophysiology of ADHD remains unclear. Attention deficit hyperactivity disorder (ADHD), one of the most commonly found neurodevelopmental disorders in children and adolescents, is characterized by impairing symptoms of inattention, hyperactivity, and impulsivity . ADHD afADHD has been associated with distinctively worse school performance independent from socioeconomic factors among children . It is aThe precise mechanisms underlying ADHD have not yet been clearly established. However, it has been shown that a reduced volume or functionality of the brain’s gray and white matter might lead to deficits of attention, cognition, processing response speed, motor planning, and other behavioral problems shown in ADHD ,16. ReceSeveral studies have measured the plasma levels of monoamines in an attempt to explicate the underlying biological mechanisms involved in ADHD . HoweverCurrently, there has been no clinically reliable biomarker for the diagnosis of ADHD, although several plausible inflammatory biomarker candidates have been suggested based on recent advances in biochemical and molecular biology . DetermiFrom this point of view, the aim of the present review article was to summarize the potential inflammatory biomarkers and the underlying mechanisms, including detailed description of the kynurenine pathway, inflammatory-cytokine-mediated regulation of the kynurenine metabolism, dysregulation of the kynurenine pathway in ADHD, and potential biomarkers in ADHD.The KP is involved in catabolic TRP degradation. It acts on glutamate receptors in the central nerve system and potentially regulates the essential mechanisms of ADHD by generating a number of neuro-active compounds collectively called kynurenines that could interact with neurotransmitter (NT) receptors in the central nerve system (CNS) . This paN-formylkynurenine [The KP pathway is initiated either by tryptophan-2,3-dioxygenase (TDO) in the liver to open the TRP indole ring, or indole-2,3-dioxygenase (IDO) in the brain to produce an instable metabolite of nurenine ,41  can regulate the formation of L-KYN in microglia, resulting in the formation of AA, 3-HK, 3-HAA, and quinolinic acid (QUIN). QUIN is excitotoxic at NMDA glutamate receptors. It has a synergistic effect with 3-HK in generating oxidative stress . In astrDuring the last decades, the underlying regulatory mechanism of kynurenine metabolism has been extensively investigated as it has a role in CNS disorders ,48,49. ATo the best of our knowledge, only three research groups have investigated serum levels of kynurenines in patients with ADHD. Evangelisti et al.  have repThe first step of TRP catabolism takes place with IDO and TDO, which are generally known to be differently modulated. While IDO is induced by pro-inflammatory cytokines during immune response, TDO is induced by glucagon and corticosteroids . There iWhile IFN- γ is regarded as the primary IDO inducer, there is evidence showing that the expression of IDO can be induced independently of IFN- γ ,57,58. IExperiments investigating the role of anti-inflammatory cytokines in IDO expression have shown limited and often conflicting results. This might be due to differences in the models used and experimental conditions applied. For instance, IL-10 as one of the major anti-inflammatory cytokines decreased LPS-mediated IDO protein expression in a dose-dependent manner. However, IFN-γ-mediated IDO protein expression was increased by IL-10 in mouse bone marrow-derived dendritic cells (BMDCs) . This inOther than those anti-inflammatory cytokines, pro-inflammatory cytokines (such as TNF-α and IL-1β) and toll-like receptor (TLR) agonist (such as LPS) can synergistically potentiate IFN-γ-treated IDO expression . MoreoveIFN-γ-independent IDO induction is supported by studies using primary mouse microglial cells demonstrating that IFN-γ mRNA is not detectable whereas IDO mRNA levels are increased after LPS stimulation ,56. OtheSimilar to IDO, pro-inflammatory stimuli may activate KMO enzymes downstream of the pathway. After the systemic inoculation of LPS, KMO expression is induced in rat brain . KMO is Compared to the expression levels of IDO and other kynurenine enzymes in the neurotoxic branch of the KP, KAT expression is neither elevated nor changed in response to pro-inflammatory stimuli. Systematic LPS inoculation of LPS causes no change in KAT II in rat brain cells . In immoCNTFR) were associated with the severity of ADHD inattentive symptom. Odell et al. [CNTFR and ADHD in both children and adults. They also reported an association between ADHD and major histocompatibility complex genes, demonstrating the role of inflammation and autoimmunity in this disorder. However, recent findings of a genome-wide association meta-analysis have failed to replicate these results [Genetic studies have supported that gene polymorphisms are linked to the inflammatory pathway in ADHD. In a total of 398 subjects, Smith et al.  evaluatel et al.  conducte results .Another genome-wide association study for 478 ADHD patients and 880 controls has suggested no significant SNPs . HoweverWhile there is a good number of genetic studies supporting the association between inflammation and ADHD, there are high variations for methodologies applied in each study. Highly heterogeneous genetic features and clinical manifestation might take part in the observed variation. Currently, there is no consensus about which inflammatory-related genes precede ADHD. Future research on populations with heterogeneous features might provide more conclusive findings.A delay in the development of cortical maturation may cause evident deficits in neuropsychological performances in ADHD . AlthougAlthough no report has directly explored cytokine and kynurenine profiles at the CNS level in ADHD, a few studies have tried to establish the association between these markers and behavioral endophenotypes by measuring their serum levels. Oades et al. demonstrated that levels of S100b are negatively associated with oppositional and conduct problems in ADHD . Their sRecently, psychiatry research studies have measured and examined how individual cytokines known to be related to inflammatory processes are related to particular diagnostic categories and related phenotypes. Individual relationships of these markers with various mental disorders in perinatal and offspring outcomes, chronic states, and pre/post-treatment have been examined based on cytokines, C-reactive protein, hormones, neurotrophins, and so on. Some commonly investigated cytokine measures are summarized in It has been reported that neuroinflammation might underlie the neurodevelopment of the immune system, resulting in changes in normal microglia, astrocytes, chemokines, cytokines, oxidative stress, and related metabolism in the first months or early years of life . In chilConsistently, mechanistic evidence has been demonstrated by animal studies, which show maternal immune activation in offspring with ADHD . During Peripheral pro-inflammatory cytokines can cross the brain through humoral and neural pathways and maintain inflammatory responses via neuroimmune systems. Inflammation-related cytokine changes in the brain are known to cause neurotransmission changes in TRP metabolism and dopaminergic pathways in the brain, similar to those seen in patients with ADHD . At thisIn recent studies, various inflammatory cytokines in the central and peripheral samples have been proposed as feasible potential biomarkers of ADHD risk. Chang et al. ,76 evaluOades et al.  investigMisiak et al.  conducteChang et al. investigated inflammatory biomarkers in ADHD and revealed that morning salivary cortisol levels were lower in youths with ADHD than in healthy controls ,76. ThisAlthough the most recent research on ADHD biomarkers has suggested the possibility of finding more objective forms of diagnostics compared to the current diagnostic criteria in clinical use today, it remains unclear how these discrete markers are associated with diverse clinical manifestations and different populations that persistently confound research on ADHD. A few observational studies have evaluated different variables without adjusting or controlling confounders to investigate the role of inflammation in the pathophysiology of ADHD, yielding conflicting results. Further research overcoming these limitations of previous research studies performed so far is needed.The goal of this review was to explore the possible underlying etiopathophysiology of KP in the regulation of ADHD. This reviewed summarized the current knowledge on the range of possible peripheral inflammatory biomarkers. Uncertainty remains as to whether neuroinflammation is a cause or consequence of the risk of developing ADHD. However, some researchers have demonstrated that the KP and subsequent neuroinflammation have a certain degree of association with the state of ADHD. Searching for reliable peripheral inflammatory biomarkers is of great interest in terms of eliminating uncertainty and overcoming diagnostic and treatment difficulties in the clinic. Although a solid neuroimmunological basis has been established through recent neuroimmunological advances, there is a need for further studies to determine how changes in inflammatory markers are related to disease status or whether these markers can be used to detect the development of ADHD and predict its progression and response to treatment.


Our gastrointestinal system functions to digest and absorb ingested food, but it is also home to trillions of microbes that change across time, nutrition, lifestyle, and disease conditions. Largely commensals, these microbes are gaining prominence with regards to how they collectively affect the function of important metabolic organs, from the adipose tissues to the endocrine pancreas to the skeletal muscle. Muscle, as the biggest utilizer of ingested glucose and an important reservoir of body proteins, is intricately linked with homeostasis, and with important anabolic and catabolic functions, respectively. Herein, we provide a brief overview of how gut microbiota may influence muscle health and how various microbes may in turn be altered during certain muscle disease states. Specifically, we discuss recent experimental and clinical evidence in support for a role of gut-muscle crosstalk and include suggested underpinning molecular mechanisms that facilitate this crosstalk in health and diseased conditions. We end with a brief perspective on how exercise and pharmacological interventions may interface with the gut-muscle axis to improve muscle mass and function. The gut consists of trillions of microbial cells, which plays an important role in many aspects of human health and can influence muscle health through dietary fiber, proteins and metabolic by-products . The gutvia the gut-muscle axis, it can create new avenues of treatment to improve muscle health through direct means such as probiotics or indirectly via dietary interventions or prebiotic supplementation. In this article, we summarize recent animal and human studies that suggest the role of gut microbiota in influencing muscle health, and on how gut microbes may in turn be altered during certain muscle disease states. Insofar, the aim is not to provide a systematic review but rather to give a perspective based on existing evidence that exercise and pharmacological interventions provide benefit by impinging on the gut-muscle axis.Muscle health is important because skeletal muscles are major sites of insulin stimulated glucose uptake, and thus play a key role in glucose homeostasis and whole-body metabolism. Furthermore, low skeletal muscle mass is often associated with frailty in older adults which increases their susceptibility to adverse outcomes and negatively affects their quality of life. It is also associated with many metabolic diseases such as Type 2 Diabetes Mellitus (T2DM). If it turns out that the gut microbiome does indeed strongly influence muscle health Sdh) gene and reduced activity of the mitochondrial SDH enzyme. The amount of mitochondrial DNA content also reduced and there was evidence of dysfunctional mitochondrial biogenesis and oxidative capacity of the soleus (oxidative) and extensor digitorium longus (glycolytic). Reduced expression of glycolytic genes was observed in these muscle groups. However, despite a possible reduction in oxidative metabolic capacity, the GF mice performed as well as SPF mice when challenged till exhaustion, suggesting the involvement of other compensatory pathways especially during the endurance phase. Importantly, upon treatment with SCFA, the muscle strength of GF mice increased as compared to the untreated GF mice, suggesting that SCFAs may be an important link between gut microbiota and muscle function. Transplantation of gut microbiota from SPF mice to GF mice helped to restore muscle mass and mitochondrial DNA content in GF mice muscle. This suggests that modulation of microbiota could potentially be used in humans as a way to treat conditions such as sarcopenia. When similar experiments were repeated in piglets, the results were largely similar to what was observed in mice  mice lacking in gut microbiota displayed reduced skeletal muscle weight, as compared to conventional or specific pathogen free (SPF) mice which have an intact gut microbiota and immune system . Upon hi in mice . The GF In another study, when three different antibiotic regimens  were administered to mice, there was a decrease in mass of the gastrocnemius-soleus complex of the mice . HoweverIt is worth noting that there are also studies the suggest negative associations between gut microbiota and whole-body lean mass. In one study when GF mice were colonized by fecal samples from age matched, conventionally raised mice, the whole-body lean mass decreased by 7–9% with a 57% increase in total body fat content. It was also associated with increased plasma leptin, fasting glucose and fasting insulin levels  either using amoxicillin or tylosin phosphate developed larger bones with increased lean and fat mass as compared to controls. It trended toward increased bone in amoxicillin-treated mice and increased fat in tylosin-treated mice  and thishe cecum .Given its highly complex and multi-dimensional nature, the microbiota that evolve with different antibiotics and FMT regimens can exert different corresponding phenotypes. While gut microbiota does have affect muscle mass and function, these discrepant findings do suggest the need for more studies to determine the causality, functionality and directionality of the microbiota and its constituent members.Firmicutes/ Bacteroidetes ratio than those with normal muscle mass  suggesting a possible link between gut microbes, inflammation and changes to muscle mass. There are cross-sectional human association studies that compare patients with cirrhosis-related sarcopenia with control subjects. The principal alteration in age-related sarcopenia and cirrhosis-related sarcopenia was a reduction in SCFA-producing bacteria. Lachnospiraceae family, consisting of Lachnospira, Fusicatenibacter, Roseburia, and Lachnoclostridium, significantly decreased in age-related sarcopenia.Patients with low muscle mass or sarcopenia, in the context of organ failure or cancer, were observed to have alterations in their gut microbiome. It was reported that patients with chronic liver disease who had lower muscle mass possessed a lower cle mass . The levAkkermansia muciniphila and butyrate-producing Ruminococcus bromii likely due to a change in diet. With increasing malnourishment, there is increased abundance of LPS-producing Ruminococcus gnavus and deceased butyrate-producing Lachnospiracae and Ruminococcaceae  biosynthesis. Amongst the residents, with increasing age, there was a reduction in mucin-degrading occaceae . In a sencreased . Such ch atrophy –15.Admittedly, metabolic dysregulation and aging are complex conditions that encompass differences in nutritional intake, digestion and assimilation, drug use treatments  and background physiology and inflammation, all of which may profoundly confound microbiome changes and muscle health. Mechanistic studies are key to elucidating how the microbiome, and its metabolites, influence muscle metabolism and survivability. Furthermore, the gut microbiota diversity may also be affected by protein intake. Briefly, in a study with professional athletes from an international rugby union squad compared against healthy male controls, there was a significant increase in gut microbiota diversity and this association also correlated with protein intake and plasma creatine kinase values . GreaterIn patients with advanced gastric cancer, cachexia was associated with intestinal barrier dysfunction  with a higher degree of bacterial translocation, as compared to patients with gastric cancer but without cachexia . Levels Prevotella copri was observed in patients with cachexia and this correlated with reduced plasma levels of postulated myogenic branched chain amino acids (BCAAs) isoleucine and leucine. Lower levels of Faecalibacterium prausnitzii, a gut bacterium with known anti-inflammatory effects  and Atrogin-1 mRNA through the arylhydrocarbon receptor (AHR) pathway, inhibiting cell proliferation and myotube formation (via the Toll-like Receptor 4 (TLR4) pathway, resulting in muscle atrophy (in vitro (Indoxyl sulfate is a gut microbiome derived uremic toxin and is known for its pro-inflammatory properties in chronic kidney disease (CKD) patients . Adminisormation . Another atrophy , 39. Thi atrophy . It was in vitro  suggestiE. coli colonization in intestinal mucosa  and celiac disease (CD) are associated with a decline in muscle function and cachexia . Studiesl mucosa . Dysbiosl tissue . Furtherfunction . This raBifidobacterium spp, R. hominis, A. muciniphilia and F. parusnitzii  decreased the senescent scores and increased muscle mass in SAMP8 mice. Furthermore, it helped to maintain muscle strength in the aged mice, as seen from the higher grip force. It also reduced age related increases in inflammation by down regulating the proinflammatory cytokine TNF- α and upregulating the anti-inflammatory cytokine IL-10. In contrast to the fall in SCFAs usually seen in aging, Lactobacillus casei Shirota helped to maintain the butyrate levels in the aged mice  when gathering evidence for this perspective, but in doing so may have inadvertently missed a few relevant papers. This remains a limitation of this piece. Nevertheless, while much remains unknown about how microbiome interacts with muscle, this emerging field of research holds promise for improving our understanding of sarcopenia and other age-related muscle loss. Information on human, and animal, gut-muscle axis are now compiled into a single table . ImportaThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.WC, WL, SW, and YA wrote the manuscript. YL, EC, GF, and YA edited the manuscript. All authors contributed to the article and approved the submitted version.


Patient selection for targeted therapy in oncology has successfully relied upon standard microscopy-based methodologies, such as single-marker brightfield chromogenic immunohistochemistry. As promising as these results are, the analysis of one protein, with few exceptions, no longer provides enough information to draw effective conclusions about the probability of treatment response. More multifaceted scientific queries have driven the development of high-throughput and high-order technologies to interrogate biomarker expression patterns and spatial interactions between cell phenotypes in the tumor microenvironment. Such multi-parameter data analysis has been historically reserved for technologies that lack the spatial context that is provided by immunohistochemistry. Over the past decade, technical developments in multiplex fluorescence immunohistochemistry and discoveries made with improving image data analysis platforms have highlighted the importance of spatial relationships between certain biomarkers in understanding a patient’s likelihood to respond to, typically, immune checkpoint inhibitors. At the same time, personalized medicine has instigated changes in both clinical trial design and its conduct in a push to make drug development and cancer treatment more efficient, precise, and economical. Precision medicine in immuno-oncology is being steered by data-driven approaches to gain insight into the tumor and its dynamic interaction with the immune system. This is particularly necessary given the rapid growth in the number of trials involving more than one immune checkpoint drug, and/or using those in combination with conventional cancer treatments. As multiplex methods, like immunofluorescence, push the boundaries of immunohistochemistry, it becomes critical to understand the foundation of this technology and how it can be deployed for use as a regulated test to identify the prospect of response from mono- and combination therapies. To that end, this work will focus on: 1) the scientific, clinical, and economic requirements for developing clinical multiplex immunofluorescence assays; 2) the attributes of the Akoya Phenoptics workflow to support predictive tests, including design principles, verification, and validation needs; 3) regulatory, safety and quality considerations; 4) application of multiplex immunohistochemistry through lab-developed-tests and regulated  In immuno-oncology (IO), there is a need for improved biomarkers to predict who will respond to treatment. While immunotherapy may lead to complete remission in some patients, average response rates continue to remain within the 20%–30% rangeMultiplexing resolves clinical problems insufficiently addressed in current diagnostic testing by leveraging multiple biomarkers simultaneously. Tissue image analysis  using mIThe limitations of chromogenic IHC methods have made the implementation of mIHC in clinical trial settings challenging, particularly when incorporating more than three biomarkers. When multiple markers are co-localized, spectral absorption characteristics of brightfield (BF) dyes typically prevent reliable unmixing for per-target quantitation . This isPrevailing higher-plex discovery platforms in cancer research that use different detection modalities can multiplex 10 s of proteins iteratively or simultaneously. However, test throughput and economics are not well suited to translational workflows and unlikely to change despite approaches being technically compelling. Instead, these methods will provide a rich pipeline of new biomarker signatures that can be converted to simpler lower-plex assay panels that are more suitable for clinical trials and translation into eventual standard-of-care .Several technical approaches exist; details are beyond the scope of this Review (though see Published academic studies routinely demonstrate that lower-plex (4–9 marker) multiplex immunofluorescence (mIF) assays might be sufficiently sensitive, practical, and affordable to see wider-spread clinical adoption. Unfortunately, research-use-only (RUO) application of mIF in translational research lacks the rigorous controls and standardization needed to support the stringent reproducibility, sensitivity, and specificity requirements of those same mIF assays for clinical trial use. A foundation of scientific discovery, rigor is particularly critical when the performance attributes undergird drug discovery or patient therapeutic choice.Integrating image analysis (IA) solutions with mIHC/mIF assay optimization and validation has seen limited regulatory guidance and fewer harmonization effects. Certain analytic limitations of a wet-lab assay can be ameliorated with IA, but algorithms are susceptible to generating erroneous data . For exaProgress to date with the application of IA, including artificial intelligence (AI) and machine/deep learning (ML) approaches, has focused mainly on BF IHC and histopathological staining , provides an alternate approach to single-section mIHC. However, analysis of individual protein markers on serial sections is not always supportive of reliable cell type classification based on co-expression and these workflows are often not amenable to high-throughput needs of clinical studies or trials, with a few exceptions e.g., .Formalin-fixed paraffin-embedded (FFPE) tissue is the most for use method for preparing and preserving specimens for use in research and therapeutic development. Data demonstrate that upfront handling and processing of FFPE specimens have a significant impact on the quality of intercellular and intracellular components to affect results of downstream assay and analyses. For IF, it is autofluorescence (AF)—endogenously found in most tissues and enhanced by formalin fixation e.g., —that conFor any spatially resolved mIF technology to be successful and provide clinically meaningful advantages over other simpler IHC approaches, awareness of these potential stumbling blocks and others placed by pre-analytical variables is important, especially as a greater number of tested biomarkers inform the test readout. Multiplex IHC, in general, also requires greater vigilance in terms of quality control of the many analyte-specific reagents (ASR) that comprise the final assay. The difficulty of finding per-target detection controls that should be incorporated into routine testing practices can reduce the likelihood of reporting errors.Automated IHC slide staining technologies coupled with visual assessment by pathologists have been readily implemented in routine clinical care settings and have historically delivered high-value medical and clinical information at a relatively low cost. However, useability issues posed by high complexity test interpretation, using intricate analytical and bioinformatical software tools, must be addressed, once they are configured and “locked-down,” particularly by platform providers. Clinical software needs to be developed from the ground up under a robust quality assurance (QA) system and reduced to essential functionality. Choice of code base, architecture, workflow, and user interface are driven by technical and operational requirements of the test. Multiplexed data acquisition pipelines must be streamlined and operationalized to ensure the intended use of multiplex technology to enable rational clinical decisions quickly and accurately with economic benefit.,Regardless, benchmark efforts are underway, steered by national societies and working groups comprised of representatives from academia and biopharmaA new Frontier of biomarker discovery based on spatial biology presents a path toward the clinic, as workflows become practical and analytically robust. Documented design principles that include validation and verification processes will ensure meaningful translation of spatially resolved multiplexing technologies such as mIF into clinical practice based on their intended uses. Modern approaches advocate for use of mIF for exploratory clinical sample analysis as a more straightforward methodology. As such, procedural integrity is key to enabling a more comprehensive and locked-down assay, analysis, and reporting strategy—under appropriate quality control (QC) processes—necessary for regulatory device submissions.A goal of companion diagnostic (CDx) development programs is simultaneous development and regulatory approval of drug and test in parallel coordinated tracks, ensuring that when the drug is approved with test indicated and required as per the drug label, the test is also readily available. These parallel development processes converge in the form of a clinical trial to validate the CDx test’s effectiveness to identify patients by the presence or absence of a biomarker and, in turn, determine therapeutic efficacy within a population of patients selected (or not) to receive a particular drug.in vitro companion diagnostic (IVD CDx) tests, including medical device clinical investigation design and Investigational Device Exemptions .Platform choice is a key decision point in setting the specifications of an assay in this prototype stage. Intellectual property needs are defined, with a clear understanding of this and other risks. As an example, the workflow might need to be compatible with common and custom laboratory information management systems. For image-based analysis, data processing workflows may also need to support remote viewing and annotation and be capable of handling the scale and size of images and data sets in a HIPAA-compliant manner , at least with respect to a predefined cutoff profile, it is not a requirement. The collective use of multiple biomarkers offers some level of flexibility during IVD CDx development that shapes performance characteristics for a specific clinical application. Multivariate tests such as mIHC can provide several quantitative outputs that support more nuanced decision making, perhaps values of multiple variables combined using an interpretation function to yield a single, patient-specific result.In IO, the process of developing a multiplex panel usually starts with biomarker selection by a scientific team comprised of immunologists, cancer biologists, and technical specialists, focused by one or more hypotheses related to the drug and/or target biology. These exploratory-use assays typically support promising hypotheses for predicting response. Once exploratory work is complete and correlations are determined empirically, the multiplex panel may be reduced to only those markers necessary to achieve an outcome: marker reduction is a statistically driven process that strikes the right balance of simplicity and predictive power.Feedback should be sought from the FDA for clinical studies that began with an assay version different from the one ultimately filed with the agency, perhaps as simple as a manufacturer change, to another using a different IVD CDx device configuration  entirely.In the former, an analytic bridging study is designed that shows earlier and current versions of the test perform identically. In the latter, as test design or manufacture has been changed significantly, the bridging study may include re-testing all samples run with the prior version of the clinical trial test with the final validated test. Such bridging strategies should be avoided as much as possible, however, where unavoidable, built thoughtfully into trial sample, consent, and test planning for accommodating sample re-testing requirements.A product concept document defines the prototype assay and analytic plan that can be used to demonstrate proof-of-concept for the technical and clinical validity of the final IVD CDx. Loosely speaking, it identifies the link between biomarker status and the clinical outcome following treatment with the investigational drug and thus confirms a proposed biomarker hypothesis. Feasibility studies focus on establishing a minimum accepted level of analytic performance that include, but are not solely limited to, precision and reproducibility, accuracy, sensitivity, and specificity.Normally, development and production activities for a prototype IVD CDx assay are first completed to a standard as required by CLIA  clearance from the FDA, in a process called single-site premarket approval , certain performance specifications still must be met that would ensure assay consistency among users, as per CLIA and CAP guidelines. A manufacturer may also seek regulatory approval to obtain IVD  further establish specificity, and health authorities often require such data.Analytic validation studies are used to demonstrate real-world test precision, or repeatability (single site/single run/single test operators) and reproducibility (multiple sites/multiple runs/multiple test operators), chiefly at the limit of detection (LOD) or at a clinical decision point. This approach confirms the capability of the test to distinguish between positive and negative samples with consistency. The cutoff defines the positive test result and infers the prototype assay may be a clinically useful treatment decision tool, which will divide the intended patient population into likely responders or non-responders to the investigational drug.The classic CDx paradigm is that cutoff values are determined from early phase  clinical studies based on retrospective data analysis after which a clinical threshold can be prospectively validated in a subsequent late stage (III) trial.Breaking with this, cutoffs are more frequently being chosen and used prospectively for patient selection in earlier phase clinical trials before they are completely validated. This approach introduces risk as trial outcome might be determined using a cutoff that may not be fully informed by earlier studies (drug development risk) or using a CDx assay that may not have been fully validated for performance around the cutoff at the time of enrollment (diagnostic risk).To the latter point, when initial clinical outcome data are available and test cutoff is defined, the validation set used to establish analytic performance for the new device includes samples below, above, and at the cutoff (each) to ensure the prototype IVD CDx test returns an accurate result. The absence of specimens with clinical outcomes would require that the cutoff selection be informed by biomarker prevalence and/or intensity measurements.One method that has proved useful in selecting cutoff values is the receiver operating characteristic (ROC) curve  Nahm, 2. SensitiReducing a set of markers to a fit-for-purpose clinical assay is a biostatistical process that might include rank ordering measured biological parameters according to individual AUC, selecting top ranked parameters, to avoid data model overfitting, and defining a scoring equation that includes selecting thresholds to identify patients with a higher likelihood of responding to therapy. This can mean that some markers in an exploratory multiplex panel not associated with top-ranked parameters are dropped from the test or from the analysis.Sufficient AUC needs to be the overarching goal, since it drives trial and drug success. Based on an internal model assessing trial success rates and health economics considerations, a reasonable AUC target for predictive tests in IO should be 0.80 or greater see also .Recent meta-analysis of anti-PD-1/PD-L1 therapy data pooled from 50+ studies spanning 10+ tumor types and 8,000+ patients used ROC and AUC to examine the predictive value of single-marker IHC (PD-L1), tumor mutational burden (TMB), gene expression profiling (GEP) and PD-L1 mIHC/mIF. The results revealed that the category including mIHC/mIF performed significantly better compared to the other three assay types . For insThough the discovery of important predictive biomarkers is largely a medical/clinical or biological problem, statistics plays a vital role in all areas of precision medicine, ranging from study design to analysis. It is fair to state that the analytic validation of multi-variate tests will require more refined study designs than for single-plex IHC assays .,,,Among other recommendations,To provide a sense of the impact of achieving more precise, selective and clinically- or economically-actionable test , includiPractices that tease out which test methods are economically actionable are poorly characterized. More exact economic assessment of mIF as a cost-effective treatment is advised and appropriate for informing decisions for where only patients with economically actionable results will receive targeted treatment .Frequently at this stage in IVD CDx device development, the various benefits of the assay/drug pairing have yet to be demonstrated, testing costs and reimbursement rates are not considered, and assay development is primarily focused on analytic performance, such as precision and reproducibility. Meanwhile, assay results are used for exploratory purposes and typically not for trial patient selection. If the assay is intended to be used for patient selection, and might pose significant risk to patient safety in the United States, then filing of an IDE is necessary and the activities supporting analytic performance must be approved to ensure no clinical investigation delay.Additionally, studies investigating the use of these candidate diagnostics for a candidate therapy must be designed to follow FDA regulations pertaining to investigational new drugs IND24. To sQ-Subs can help coordinate interactions among FDAs different centers [for Drug Evaluation and Research CDER26, for For mBF and mIF, as automation will almost heavily underpin the analytical robustness of the final device, the performance of the assay on a specific instrument must be described in detail, including operational specifications and calibration. As CDx assays are viewed by regulatory authorities as integrated, fixed systems with strictly defined component. For example, a specific autostainer model and version(s) of operational/analytic software also become part of the device and are collectively locked-down by version control to prevent changes to the device.Device labeling, normally discussed as part of the Q-Sub process, determines how the CDx should ultimately be used to support the indicated uses with single or multiple drug or biological products. The intended use statement drives all subsequent performance attributes covered under the verification process. Any change from the established system components or parameters as described in the product package insert would be considered a deviation from the approved use of the assay and would thereby invalidate the test results.In transitioning from prototype assay to IVD product, subsequent work is performed in a controlled manner under Good Manufacturing Processes GMP30 also 21 CFR 820.30 covers design transfer to ensure device design and design outputs are verified as (transferred into) suitable production specifications. Product attributes include QC and performance specifications for raw materials, manufacturing process validation, in-process and final release QC, requirements for shipping conditions and stability determinations of raw materials, intermediate batches, and the final product(s).ISO 13485:2016 certification, specifically and particularly, ensures the design, development, production, distribution, and installation of medical devices, such as software or instruments, meets industry requirements in the international market since different countries might have different quality standards.In either instance, all verification activities are properly documented in a design history file (DHF) using a set of design input requirements that match design output specifications. Inherent to the process is the requirement for periodic design review stages when the device’s performance is assessed against the requirements, although the subsequent analytical verification of the assay provides objective evidence that the design specifications have been met according to the intended use.Key performance attributes being assessed during verification include analytical sensitivity, analytical specificity, precision, robustness, and stability.Not covered previously, robustness studies (flex- or guard-band studies) assess the ability of the IVD CDx to remain unaffected by small and deliberate changes to assay parameters. These provide an indication of its reliability to function correctly under varying conditions of proper and improper use, ensuring that slight variations encountered in the operation of the test during normal use do not affect results.Notably, key attributes of a well-designed product concept document will also include understanding and verification of the inherent  variability in sample collection and handling methods, processing, and stability. For IHC tests particularly, there is a strong appreciation for the impact of preanalytical variables  on technical aspects of testing to ensure analytical trueness.Particularly for FFPE biopsy samples, the quality of the sample is a major determinant for test success, so ensuring the assay is robust enough for low-input test material is important, provided there is established QC criteria for identifying potentially degraded samples . An addeBeyond only a few biomarkers  FDA-cleared algorithms for HER2A newly described and innovative multidisciplinary approach, called AstroPath at the Johns Hopkins University  Figure  leverageThe approach delivers on two important translational goals—creating an imaging and data analysis pipeline that is highly reproducible and accurate for classifying critical tell-tale cells in the TME, while simultaneously storing and making accessible for data mining vast data sets that come out of studies. The database is highly optimized for rapid data querying and integrated with a cloud-based digital pathology interface.® workflow of Akoya BiosciencesAstroPath, based on the PhenoImagerThe PhenoImager solution from Akoya Biosciences is a complete end-to-end workflow that supports research and clinical trial objectives for mIF . The tecThe workflow utilizes tyramide signal amplification (TSA) to intensify and stabilize fluorescent signals e.g., . DiffereA typical TSA-based workflow requires sequential detection of primary antibodies, with heat, pH or other methods  used to strip primary and secondary complexes from the tissue section, leaving the covalently-bound fluorescent tyramides at the site (labeling radius ∼200 nm) of primary antibody binding. While TSA-based workflows are iterative and therefore multi-step, with potentially 12–18 h run times, repeated exposure of tissue to high temperature or pH conditions (or other) are a consideration for occasional more-labile antigens. But this issue is readily addressed through determining marker staining order. In fact, repeated heating steps are useful to achieve more complete retrieval of many important functional markers, such as PD-L1 and Ki67.Tyramide conjugation is used by multiple platforms, including for BF mIHC using TSA-chromogens that are HRP substrates with narrow spectral signatures . DespiteThe most effective utilization of mBF has been when targets are each expressed on different cell types or with different subcellular localizations in one cell type , therebyOther benefits of utilizing a TSA mIF workflow are a flexible target detection strategy and automated IHC workflow that can be easily implemented in routine clinical care settings, even in those with modest technical resources . CurrentThe PhenoImager workflow using a multispectral imaging (MSI) system resolves each IF marker into accurate single component images by capturing spectrally resolved information at each wavelength within each pixel of an image. The most current iteration of the MSI system is the PhenoImager HT (formerly known as Vectra Polaris), paired with two visualization software programs: Phenochart and inForm.Phenochart is a whole slide contextual viewer enabling annotation of BF and FL slides scanned on the PhenoImager HT for MSI capture as FOV or WSI. This program has a live unmixing preview of WSI to support assay development and confirm staining performance. In addition, simulated DAB views of each FL channel can be provided, as can overlay of histological stains such as H&E. While mIF is visually stunning, most classically trained pathologists are unaccustomed to reviewing IF images and interpretating anatomical and morphological features in this format.Further, H&E severely limits the further use of the same tissue section for IHC purposes. Creating a simulated pathology view with removable fluorescent dyes that accurately mimic traditional BF H&E to enable section re-use with no decrease in mIF performanceinForm is a software analysis package that integrates MSI and IA to spectrally unmix and isolate multiple FL signals and remove background AF . The sofinForm output data can be imported into a variety of other IA platforms for quantitative evaluation  medicine, Health Management Organizations (HMO)/Insurers remain under significant pressure to address testing costs. For example, HMO reimbursement rates for multiplexed testsRecently, a six-center inter-site comparison study with participants from biopharma and academia was conducted to demonstrate analytical performance across multiple institutions of a translational mIF workflow. The study was termed the multi-institutional TSA-amplified mIF reproducibility evaluation (MITRE)  Figure .Based on the PhenoImager HT platform, MITRE demonstrated the analytical reproducibility within and across all sites of an integrated workflow system for a mIF assay focused on the PD-1/PD-L1 axis also including CD8, CD68, FoxP3, and CK.Further, the image analysis platform used in the MITRE study employed an AI/ML-based tool for segmenting and phenotyping cells, one which could be locked-down and shared to minimize inconsistencies. The locked-down capability also reduces discrepancies introduced by potentially varied practices related to local lab image analysis algorithm adjustments. Currently, effective interpretations of increasingly large datasets remain challenging, and analysis of such complex datasets has outstripped cognitive capacity.The push toward precision and personalized medicine has piqued interest in AI/ML approaches that structure, integrate, and interpret large datasets, to identify and validate highly predictive biomarkers to aid in clinical decision-making. This could ultimately lead to potential payoffs for patients and drug development programs. AI/ML approaches promise to parameterize and simplify vast tissue-based cellular data, consisting of protein co-expressions and spatial parameters, and to reveal easily interpretable models, such as SPS.Tumor and immune/stromal cells comprising the TME represent a continually evolving ecosystem, characteristics of which are spatial and functional intra-tumoral heterogeneity. In cancer research, a recent explosion of ultrahigh-plexing biomarker labelling and imaging modalities probe the TME with 10s of biomarkers at cellular and subcellular resolution . AnalysiFor high-dimensional single-cell phenotyping data, analysis methods that efficiently incorporate the inherent multi-parametric characteristics of such immunological data sets have been employed by the flow cytometry community for decades. Nevertheless, advantages of deploying such data processing tools within the histopathological space come with drawbacks, particularly the use of dimensionality reduction practices , moderate (Class II), or high risk (Class III). Class III devices pose the greatest risk and typically require submission of an application for premarket approval as these are For FDA Class III devices, test results are preferably compared to an established truth, a so-called “gold standard,” with the intention that a measure of accuracy is linked to the clinical utility of the test. An accurate prediction of clinical response provides clinical utility data for both the drug and device.de novo process has also been used by FDA to down-classify diagnostics that are considered “complementary” rather than “companion,” which would have previously been considered a higher-risk device.Upon establishment of clinical relevance, a diagnostic might also be submitted without a PMA application for clearance in case of demonstration of equivalence or superiority to an approved marketed “predicate device” through submission of a 510(k) applicationIVDs used to identify new target analytes that potentially hold clinical significance are considered Class III if the diagnostic information thus obtained cannot be identified by conventional methods. For example, consider a cell phenotype that cannot be confirmed using a single IHC marker or approach, mBF or mIF, that provides more clinically useful treatment decisions than traditional single-marker chromogenic IHC.Examples of lower-risk Class I IVDs are panels of single-marker IHC tests, used typically as an adjunctive to pathological diagnosis  to answeIn the United States, technologies such as scanners and software manufactured for digital pathology are considered medical devices and are regulated by the FDA. In 2017, the FDA approved the first digital pathology system for primary diagnosis using images of H&E-stained slides called the Philips IntelliSite Pathology Solution . SubsequThe COVID-19 pandemic is a likely catalyst for more pathologists to adopt a digital workflow , as manyWith regards to the regulatory evaluation of a digital WSI system, the FDA provides nascent guidance on the technical performance assessment data at the component levelFurther, the FDA recently released a position paper on AI/ML systems that structure, integrate, and interpret large datasets to aid in clinical decision-making. The “Proposed Regulatory Framework for Modifications to AI/ML-Based Software as a Medical Device,”Digital Imaging and Communications in Medicine DICOM53 is thAs expected, significant differences in data characteristics of histopathological slides scanned with different WSI systems become evident, which pose a particular issue for AI/ML when the algorithm derived with a training set or supervised model of learning does not perform well on real-world unseen or unsupervised data sets. Several international computational pathology challenges  and working/reportable ranges  to establish assay analytical sensitivity—LOD should not change when new reagent lots are used in the test, since LOD typically represents the threshold separating a positive result for an analyte from one that is negative.The inability to generate analytic response curves for commonly used clinical IHC tests in part or whole complicates the standardization of test results, because calibrators are used to establish variability between and within laboratories, and for operational qualification of new lots of assay components.With mIHC, there is minimal standardization of antigen- or reporter-specific calibrators or batch-run controls. Like the subsequent steps that comprise the analytic phase of the test, proper antigen retrieval is not usually verified with the benefit of an external control; distinguishing between staining variability caused by antigen retrieval  or alterations in staining reagents or procedure  is an identified area that needs addressed for clinical deployment of mIHC. Efforts are underway to develop and validate batch run controls using cell pellets and/or curated tissue microarrays. Nonetheless, these approaches have been more suited to visual review  sensor found in most microscope cameras. RGB sensors lack the spectral resolution to accurately unmix, or de-convolute component spectra, for independent quantitation. In recent years, new BF colors with chemistry based on FL dyes have become available that form new colors when combined . The useFor mIF, quantitation is based on isolating marker fluorophore emissions from one another, either by purely optical means, or by implementing spectral unmixing techniques which is needed for accurate quantitation of more than four co-localized markers. In such circumstances, spectral unmixing compensates for channel crosstalk and support accurate quantitation, like detector spillover compensation methods in flow cytometry, but performed on a per-pixel basis.When using FFPE for mIF, MSI also isolates marker fluorophore emissions from AF. Unmixing spectral libraries are developed using control tissues stained with one fluorophore at a time, including an unstained slide to provide an AF spectrum.Multispectral imaging is the first step toward reliable compensation-based quantitative measurement of mIF and, more broadly, significantly improves the ability of mIF assays to be effectively quantified by IA platforms since the overall signals within a given pixel can be partitioned correctly into its different FL components. MSI alleviates the major challenge associated with IF and FFPE samples: reduction of the impact of AF, which is particularly important for fluorophores at the blue/green end of the visible spectrum . MSI canAnother challenge to standardization in IHC is the so-called “reproducibility crisis”Experiences documented by Nordic Immunohistochemical Quality Control NordiQC67, one Unfortunately, such national programs or umbrella consortiums , tumor annotation or FOV selection, and results review and sign-off.Laboratory Developed Tests, LDTs, have been a cost-effective way to bring forward complex technology or multiparameter data analysis needs into the diagnostic space, with regulatory oversight provided by CLIA and CAP rather than the FDA. LDTs analytically validated to be CLIA-certified and/or in compliance with CAP-guidelines are typically viewed as necessary for inclusion in drug trials to support primary or secondary endpoints, and to provide reliable data to support consideration for moving a test toward patient selection and eventual CDx use.FDA officially holds discretionary authority to regulate LDTs under the Public Health Service Act, and has published guidelinesASRs are key components of LDTs and some other diagnostic tests, subject to GMP and defined under 21 CFR 864LDTs, as a locked device or platform, are comprised of a set of reagents , sample preparation, instrumentation, software, and test scoring/interpretation guidelines that are validated together. The laboratory provider for clinical trial testing acts as an LDT manufacturer. Traditional IVD CDx are developed by a conventional IHC diagnostic manufacturer . Simultaneously, these machines are also burdened by high cost, maintenance complexity, and low throughput.A recent example of an LDT where spatially resolved technology has been included in clinical decision-making at the local level is PROFILESeveral other LDTs that are multianalyte assays with algorithmic analyses (MAAAs) incorporate results from a panel of tests, with or without other clinical information, into an algorithm to generate a risk or probability score. These have diagnostic superiority in diseases in which single biomarkers have limited validity or a non-invasive biomarker is lacking.ProMarkTissueCypherLDTs can only be run in a single laboratory which poses a significant restriction to the global testing footprint and makes LDTs less appealing for biopharma because of its impedance to drug adoption and use. Excitingly, the current trend for IO is that Phase IIb are being used as pivotal or registrational trials, and thus smaller regional studies are more frequently including patient pre-qualification that would benefit from novel technology use.Single-site PMAs (ssPMA) offer a practical interim step between LDT CDx and distributed IVD CDx, when the assays depend on new and complex methods and technology, such as with mIF. ssPMAs are essentially LDTs that are cleared through the FDA process, which requires analytical validation aligned with standard PMAs for distributed IVDs and Class III medical devices but reduced in scope because the test is run at only one site.As with distributed IVD CDx, ssPMAs require post-market surveillance of assay performance  and a proactive and systematic risk-benefit process that gathers and analyzes quality, performance, and safety data; few non-IVD manufacturers have experienced this. Concomitantly, only a few LDT PMAs have been filed as IVDs and cleared for approval by the FDA.via the PMA process utilizing centralized testing at a single site and, as such, often include the use of highly complex technologies or technological advances. Nevertheless, ssPMA may also follow EAP special designation for approval if meeting unmet medical needs or providing clinically meaningful advantages over other on-market devices.LDTs are an attractive strategy for fast market entry In Vitro Diagnostic Devices Directive 98/79/EC  are currently CE-IVD labeled under IVDD. Under IVDR and resembling current FDA approval standards, a performance evaluation will be required, including a scientific validity report and analytical and clinical performance data. To date, only two systems - the Philips IntelliSite Digital Pathology Solution  and LeicIn Vitro Clinical Test (IVCT). The VALID Act proposes a risk-based framework for IVCT regulation. High-risk novel tests require approval requirements comparable to existing medical device regulations, while lower and moderate-risk tests might go to market after passing through technological certifications. Unlike IVDR, the VALID Act would grandfather existing LDTs in clinical use  or by assessing physiological or anatomical patient characteristics that might affect response.Maturing as an analytical platform, mIF provides reliable and reproducible results that may be generated in a clinically suitable, high-throughput workflow. The spatial biology approach provides several advantages over other biomarker modalities by enabling deeper interrogation of the intricate cellular- and protein-level co-expression, localization, and arrangement within the TME.Increasingly sophisticated data analysis tools have facilitated the identification of a subset or subsets of biomarkers using a SPS which can predict a disease state, determine the likelihood of disease progression, or calculate the probability of responding to therapy , 7. MultImmune checkpoint inhibitor therapy has clearly demonstrated efficacy for subsets of late-stage patients, and the range of therapeutic options is rapidly expanding, with greater than 80 FDA approvals involving anti-PD-1/PD-L1 antibodies since 2014For example, a choice between administering anti-PD-1/PD-L1 monotherapy, which can be very effective by itself, and combining it with other ICIs  or chemotherapy has material implications for the patient. Albeit a genetic test, Exact Science’s Oncotype Dxin situ (DCIS) and triple negative breast cancerAn emerging need for mIHC is in the neoadjuvant and adjuvant settings, where trials are showing promise but: patient populations are substantially larger than in the metastatic setting, surgery alone often can cure, and the health system is not capable of supporting broad application of these expensive therapies. Cases in point are recent trial results from intralesional injection of ICIs, for ductal carcinoma Despite many advantages, substantial workflow and infrastructure challenges still act as barriers to adoption that will need to be addressed before mIHC (mBF and mIF) can fully enter clinical practice. That said, while spatial biology involving mIF has mostly been applied toward research applications to date and early-phase exploratory trials, there are signs of progress towards clinical utility or later stage clinical trial use . Approvahttps://clinicaltrials.gov/ct2/show/NCT05548296) and, if it accurately establishes therapy benefit, will be an FDA-approved IVD for a kinase inhibitor of DNA damage response.One example of this is the OncoSignature™ mIF test under development by Akoya in collaboration with Acrivon TherapeuticsThe prevalent model of technology adoption as well as the ability to integrate treatment information from mIF will determine the future relationship of this modality with that of the clinical pathologist . Neverth


The intestinal microbiota is a community of microorganisms that subsists within the gastrointestinal ecosystem. In human health, the role of the gastrointestinal microbiota is to maintain a dynamic balance with the host. This balance plays both local and remote roles in critical physiological processes, particularly inflammation, and the immune response .Natural polysaccharides are polymeric carbohydrate macromolecules and sources of fermentable dietary fiber. Polysaccharides are the most abundant dietary components in the gut microbiota and are deeply involved in host health . EmerginThis special issue includes eight papers, seven of them are original publications ,9,10,11,For instance, using a rat model of liver transplantation in steatotic and non-steatotic livers from donors after brain death, Micó-Carnero et al. (2021)  found thIn animals, but in this instance in a mouse model of metabolic syndrome, Ejima et al. (2021)  showed tPrevotella-dominant enterotype may have a more pronounced benefit on insulin resistance markers upon consumption of hemicellulose-rich foods [In humans, studies included in this special issue cover different stages in life. In a study conducted on Mexican children and adults, Martínez-Medina et al. (2021)  examinedch foods .Fiber intake also induced changes in a population of Spanish children who consumed infant cereals differing in whole grain and sugar content as first weaning foods. Results from this study indicated a supporting effect of infant cereals with 50% whole grains and a reduced sugar content over infant cereals manufactured with refined hydrolyzed flours on the infant microbiota .The fact that diet is a determinant of bodyweight and gut microbiota composition was supported by the results reported by Rodríguez-Lara et al. (2022) . In thisBrassica rapa L. extract. The oral administration of this vegetable could be responsible for modulating the gut microbiota to increase fiber-degrading bacteria and butyrate-producing bacteria [Another intervention study was conducted on healthy adults and mice fed with fermented bacteria .Under the hypothesis that fine differences in carbohydrate linkage structure would govern microbial community structure and function independently of variation in glycosyl residue composition, Romero-Marcia et al. (2021)  fermenteFinally, the review included in this collection presents a summary of recent knowledge regarding how dietary polysaccharides affect gut microbiota composition and host health. The immunomodulatory properties of certain polysaccharides are crucial to the regulation of immune responses during the progression of certain diseases . It is bThe present special issue could be considered a comprehensive summary of the ongoing research into polysaccharides/fibers as modulators of gut microbiota. It is noteworthy that these data highlight the crucial role played by polysaccharides in host–gut microbiota composition and function. Thus, intestinal fiber fermentation is a highly effective tool for modulating microbiota, its functionality, and inflammation.