Despite improvements in medical science and public health, mortality of community-acquired pneumonia (CAP) has barely changed throughout the last 15 years. The current SARS-CoV-2 pandemic has once again highlighted the central importance of acute respiratory infections to human health. The “network of excellence on Community Acquired Pneumonia” (CAPNETZ) hosts the most comprehensive CAP database worldwide including more than 12,000 patients. CAPNETZ connects physicians, microbiologists, virologists, epidemiologists, and computer scientists throughout Europe. Our aim was to summarize the current situation in CAP research and identify the most pressing unmet needs in CAP research.To identify areas of future CAP research, CAPNETZ followed a multiple-step procedure. First, research members of CAPNETZ were individually asked to identify unmet needs. Second, the top 100 experts in the field of CAP research were asked for their insights about the unmet needs in CAP (Delphi approach). Third, internal and external experts discussed unmet needs in CAP at a scientific retreat.Eleven topics for future CAP research were identified: detection of causative pathogens, next generation sequencing for antimicrobial treatment guidance, imaging diagnostics, biomarkers, risk stratification, antiviral and antibiotic treatment, adjunctive therapy, vaccines and prevention, systemic and local immune response, comorbidities, and long-term cardio-vascular complications.Pneumonia is a complex disease where the interplay between pathogens, immune system and comorbidities not only impose an immediate risk of mortality but also affect the patients’ risk of developing comorbidities as well as mortality for up to a decade after pneumonia has resolved. Our review of unmet needs in CAP research has shown that there are still major shortcomings in our knowledge of CAP. In 1918, Sir William Osler observed that pneumonia had replaced tuberculosis as the leading cause of death in Europe and described pneumonia as the ‘‘Captain of the men of death’’ [Even one century later, pneumonia remains a major health concern and lower respiratory tract infections continue to be the leading infectious cause of death with more than 2.3 million deaths and more than 91 million years of life lost in 2016 [Pneumonia is defined as an acute infection of the pulmonary parenchyma and it is commonly classified by the “pneumonia triad” into community-acquired pneumonia (CAP) as the most common form of acquisition, hospital-acquired pneumonia (HAP) including ventilator-associated pneumonia (VAP) and pneumonia in the immunocompromised host , 4. The The annual incidence of CAP requiring hospitalization ranges from 1.1 to 8.9 per 1000 inhabitants  and the In 1999, the German Ministry of Education and Research published a call to initiate an excellence competition for competence centres in infectious diseases. Through this call the “network of excellence on Community Acquired Pneumonia” (CAPNETZ) was founded in 2001 . CAPNETZTo discuss and identify which future research areas should have the highest priority in adult CAP research, CAPNETZ invited both internal and external CAP experts to participate in a 2-day scientific retreat. This paper summarizes the main research topics discussed, and highlights areas of future research, which deserve high priority to improve the understanding of CAP in order to develop novel prophylactic and therapeutic measures and ultimately reduce the burden of CAP.The identification of unmet needs in CAP research followed a four-step procedure as depicted in Fig. Since the SARS-CoV-2 pandemic emerged after the scientific retreat was held no unmet needs where identified prior to the meeting. However, due to the significant impact of the SARS-CoV-2 pandemic, sections on the Covid-19 disease have been added post hoc.Defining the causative pathogen in patients with pneumonia is relevant for ensuring the best-suited antimicrobial treatment. However, microbial diagnosis of pneumonia is still based on culture in most hospitals worldwide and a causative pathogen is only detected in the minority of patients. In a recent CAPNETZ study, bacteraemia rates in CAP were 0.5% in outpatients, 7.8% in hospitalized patients, 12.4% in patients admitted to an intensive care unit (ICU), and a relevant proportion of patients with bacteraemia and CAP (34.6%) was afebrile . With thDefine an improved gold standard for microbial diagnosis of community-acquired pneumonia: Currently, microscopy and culture of lower respiratory tract specimens as well as blood cultures, detection of urine antigen detection and serology are routinely used to complement thorax imaging results in diagnosing pneumonia. However, most tests (particularly serology) lack sensitivity and specificity, while tests with a high sensitivity, such as PCR for non-influenza respiratory viruses may currently not properly translate into clinical relevance , 35. No With the emergence of resistant or multidrug resistant (MDR) pathogens and increasing knowledge about potentially harmful or protective microbiomes , antibioMetagenomics offers a potential solution. Most studies utilizing “microbiome” or metagenomics techniques to date have used target gene approaches such as sequencing amplicons of the 16s rRNA subunit . As a moMetagenomic research has, to date, been conducted in relatively small studies in limited geographical locations . Much laStudies show that molecular techniques out-perform culture in identification of bacterial pathogens , 42, butClinicians are not bioinformatics-scientists. Therefore, automated informatics tools that can rapidly analyse sequencing data and provide results to clinicians in a usable format are required. Antibiotic resistance genes may be detected by sequencing but their relationship to phenotypic resistance in bacteria is not always known, particularly in Gram-negatives.versus non-infectious differential diagnoses. Different methods are available with chest radiography being the most abundantly used [The primary role of imaging in CAP is to confirm the clinical diagnosis. The major diagnostic challenges are the correct identification of opacities and consolidations as being evoked by infectious tly used . Howevertly used , 45. Comtly used –48. Due tly used , 49. Furtly used .Pathogen prediction through imaging analysis: Although radiographic findings do currently not allow the diagnosis of a specific pathogen, a differential diagnosis may be possible using radiological pattern recognition. Deep learning approaches may help to identify patterns that are indicative of specific viral or bacterial CAP. Integrating clinical data available at time of diagnosis may further improve such an approach.Pre-test probability for general practitioners for chest radiography: Although chest radiographs are routinely used, the efficacy as a diagnostic tool has not been determined. The reduction of unnecessary chest radiographs particularly in the outpatient setting would decrease healthcare costs, and radiation exposure. However, even in the “Prospective Cough Complication Cohort Study (3C)”, where patients with acute cough received chest radiographs, the positive predictive value of at least one of four clinical signs of CAP  for a radiography-confirmed pneumonia was low at around 20% . In anotBiomarkers may be an effective way to identify patients with severe infection and to monitor and eventually predict the course of infection. In CAP, biomarkers may be indicators of inflammation , may be released specifically after lung injury due to infection or originate from the genome of either the host or the pathogens. Numerous biomarkers have been investigated including pro-atrial natriuretic peptide , pro-vasAs the research on immune-modulatory therapies intensifies, so will the search for the appropriate biomarkers for severity and treatment response . HoweverIdentifying low risk-patients that can be treated in an outpatient setting enhances patient satisfaction and reduces costs. CRB-65 is frequently used as a first step but should be supplemented by oxygenation status, assessment of instable comorbidities and functional parameters . IdentifHarmonization with sepsis risk stratification and implementation of prediction tools: Sepsis is a major complication in CAP. Current sepsis recommendations employ a risk stratification strategy with similarities to CAP risk stratification by using a simple screening score  and a more specific high-risk prediction score (SOFA) . HoweverIndividualization of management-based risk stratification: Today, medicine is heading towards the broad application of state-of-the-art medical technologies including “omics” approaches, which might allow more tailored treatment approaches based on a better understanding of a patient’s individual state of health . In addiRisk factors for and management strategies against early post-discharge complications: Studies show high re-admission or even early death rate after discharge from hospital because of CAP , 76. So Stratification strategy for the coverage of multidrug resistant pathogens: In Europe, MDR pathogens are rare in CAP, but still can complicate the choice of empiric antibiotic therapies and may result in poor outcomes . HoweverThe early initiation of antibiotic therapy has been shown to provide a survival benefit . TreatmeTreatment guidelines differ for outpatients and only little information is available on CAP-outpatients. In the hospital, either antibiotic monotherapy or combination therapy is applied but up to now no significant differences in outcome apart from adverse effects have been observed . Most anViral pathogens are increasingly recognized as a cause of pneumonia, especially among immunocompromised patients. Although more than 20 viruses have been linked with CAP, antiviral drugs were only available for the treatment of influenza or respiratory syncytial virus (RSV) pneumonia  before CSecondary bacterial pneumonia is frequently observed in viral pneumonia , 85 and M. pneumoniae infections throughout the world [Monitoring of ß-lactam antibiotic and macrolide antibiotic treatment and development of resistance: ß-lactams have been the “antibiotic backbone” of antimicrobial therapy of pneumonia for decades . Howeverhe world –91. Antihe world .Development of new antibiotics vs. improved usage of available antibiotics: Although the number of newly approved antibiotics has tripled in the past 6 years after a 90% decrease between 1983 and 2012, MDR bacteria pose an increasing threat . Mis- anOverview of the treatment of CAP in an outpatient setting: A high proportion of CAP patients is treated in an outpatient setting , 70. A cAn excessive inflammatory response seems to be partly responsible for treatment failure in some patients and has been associated with poor clinical outcomes . DiffereFurther, due to the hyperinflammation state seen in some patients with Covid-19 several immunomodulatory agents, with the intend to block the inflammatory pathway, have been evaluated , 99. FarAs the understanding of the underlying pathophysiology improves, specifically tailored agents for immunomodulatory therapy will likely help to avoid adverse outcomes for specific patient groups.Identification of patients that benefit most from macrolides: In addition to antimicrobial effects, macrolides also have an immunomodulatory effect , 106. SeA recent CAPNETZ study has used a machine learning approach to identify patients who benefit most from macrolide treatment . Such a Immunoglobulins: Immunoglobulins have been proposed as a promising adjunctive therapy option for severe sepsis, but have only been investigated in small studies . Both, IPathogen-directed strategies: In addition to antibiotic treatment, a pathogen-directed strategy may include blockers of pathogenicity, virulence, or toxins. These may be small molecule inhibitors or monoclonal antibodies . AdditioHost-directed strategies: Alternative strategies comprise of specifically stimulating early local immune responses against pathogens, dampening particular components of the local and systemic immune responses to avoid tissue injury, increasing tissue resilience and improving resolution of inflammation and tissue repair.Vaccines against pneumococci and influenza virus, the most frequent bacterial and viral causes of CAP, are available. Influenza vaccination has been shown to reduce the number of severe CAP cases and improved overall long-term survival in patients with CAP during influenza seasons . The knoRecently, a quadrivalent influenza vaccine that includes both influenza B lines, i.e. Yamagata and Victoria, has been made available for clinical use . It has Two types of pneumococcal vaccines are used: pneumococcal conjugate vaccines (PCVs) and pneumococcal polysaccharide vaccines (PPVs). Although PPV has been shown to prevent pneumococcal bacteraemia, its protective effect against non-invasive pneumococcal pneumonia and its effectiveness in the immune-compromised host are limited –128. InfOptimal use for pneumococcal polysaccharide vaccines and pneumococcal conjugate vaccines: Although the two vaccines PPV23 and PCV13 share some serotypes they generate different immune responses . While bNovel vaccines: Like a universal influenza vaccine, a serotype independent pneumococcal vaccine would decrease the global CAP burden tremendously. Currently, a 15-valent PCV was announced in the US and a 20-valent PCV is close to market license . HoweverThe progress in microbiological diagnostics—and also the recent SARS-CoV-2 pandemics- has uncovered the burden of non-influenza respiratory viruses in CAP. Particularly, RSV seems to expose a substantial burden on adult CAP. Therefore, a RSV vaccine is highly desirable but remains a challenge , since eIn order to facilitate a rapid and efficient immune response against invading microbial pathogens, the lung combines different defence strategies including anatomic, mechanical, humoral, and cellular mechanisms aiming towards the rapid expulsion of pathogens . The lunDetermination of different inflammatory phenotypes for personalized medicine: Despite sharing the same underlying pathogen, in some patients CAP manifests as a serious disease while in others the course of disease is mild. The susceptibility to infection as well as CAP severity is most likely a phenotypical trait determined by uncountable pathogen- and host-specific factors, including polymorphisms in many collaborating genes in otherwise healthy persons , immunosPulmonary long-term consequences of CAP: While most patients return to normal lung function following pneumonia within weeks to months, some fail to recover ad integrum due to pleura-involvement or parenchymal alteration, and some may be at an increased risk of developing chronic non-infectious lung inflammation including cryptogenic organizing pneumonia (COP) and idiopathic pulmonary fibrosis (IPF) after an episode of pneumonia. The early identification of these patients would allow taking timely countermeasures. For this, specific markers  and therapeutic strategies need to be identified. Therefore, a deeper patho-mechanistic understanding of the pulmonary long-term sequelae of CAP is needed.Extra-pulmonary long-term consequences of CAP : Patients suffering from pre-existing lung diseases such as asthma or COPD are often treated with either inhaled or systemic corticosteroids . AlthougInfluence of CAP on dementia and neurological damage: As in sepsis, during the acute phase of pneumonia, confusion is frequently observed , 159. EvHospitalized CAP patients have an increased risk of major acute and long-term cardiovascular complications  , 160, whSpectrum, incidence and outcome of cardio-vascular disease after pneumonia: While there is clearly emerging evidence from epidemiological and preclinical studies for the association of pneumonia with both short- and long-term cardiovascular events, a comprehensive analysis of the various clinical manifestations  of cardiovascular disease (CVD), their incidence at different time points following a pneumonia event, and their association with outcome is as yet lacking, but would be critical to develop better diagnostic tools and ultimately tailor interventional trials.Patients at increased risk of developing cardio-vascular disease after pneumonia: Patients with pre-existing chronic CVD have the strongest risk, followed by patients with common comorbidities including chronic obstructive pulmonary disease (COPD), ischemic heart disease, and diabetes . The infS. pneumoniae into the myocardium with formation of microscopic lesions [Prevention of cardio-vascular disease after pneumonia: At present, our understanding of the patho-mechanisms that drive acute and chronic CVD following pneumonia is at best rudimentary. Discussed mechanisms include but are not limited to invasion of microbes such as  lesions , or diss lesions . A detaiPneumonia has been known as the leading infectious cause of death for more than a century and many attempts have been made to change this fact. However, the mortality of CAP has barely changed in the last 50 years and the Covid-19 pandemic has once again highlighted the central importance of acute respiratory infections to human health. Pneumonia is not just an infection of the lung but a complex disease where the interplay between the pathogen(s), immune system and comorbidities not only impose an immediate risk of mortality but also affect the patients’ risk of developing comorbidities as well as mortality for up to a decade after the pneumonia has resolved. Despite the importance of pneumonia on human health and the fact that many of the identified topics have been focus points for several years, our review of unmet needs in CAP research has shown that there are still major shortcomings in our knowledge of CAP. The poor evidence base that exists for most clinical decisions in acute respiratory infections can no longer be considered acceptable and a co-ordinated focus and investment into research on acute respiratory infections is now needed.Unmet needs have been identified for diagnostics, risk stratification, treatment, adjunctive therapy, and prevention. Major knowledge gaps include immune response, role of comorbidities, and long-term cardio-vascular complications.


Skeletal muscle dysfunction (SMD) is a prevalent extrapulmonary complication and a significant independent prognostic factor in patients with chronic obstructive pulmonary disease (COPD). Mitochondrial dysfunction is one of the core factors that damage structure and function in COPD skeletal muscle and is closely related to smoke exposure, hypoxia, and insufficient physical activity. The currently known phenotypes of mitochondrial dysfunction are reduced mitochondrial content and biogenesis, impaired activity of mitochondrial respiratory chain complexes, and increased mitochondrial reactive oxygen species production. Significant progress has been made in research on physical therapy (PT), which has broad prospects for treating the abovementioned potential mitochondrial-function changes in COPD skeletal muscle. In terms of specific types of PT, exercise therapy can directly act on mitochondria and improve COPD SMD by increasing mitochondrial density, regulating mitochondrial biogenesis, upregulating mitochondrial respiratory function, and reducing oxidative stress. However, improvements in mitochondrial-dysfunction phenotype in COPD skeletal muscle due to different exercise strategies are not entirely consistent. Therefore, based on the elucidation of this phenotype, in this study, we analyzed the effect of exercise on mitochondrial dysfunction in COPD skeletal muscle and the regulatory mechanism thereof. We also provided a theoretical basis for exercise programs to rehabilitate this condition. Chronic obstructive pulmonary disease (COPD) is a commonly recognized respiratory disease characterized by persistent and progressive expiratory-airflow limitation . HoweverBecause COPD SMD has multiple adverse consequences, exploring the underlying mechanism of SMD can help improve patients’ survival status. Mitochondrial-phenotype changes are a crucial link in skeletal muscle’s structural and functional impairment in COPD . StructuIn recent years, the role of physical therapy (PT) in the management of COPD has become prominent . As an e2+ storage and release, and generation and scavenging of oxidative substances [Mitochondria are double-membrane organelles that undertake the tasks of cell material synthesis, energy conversion, and information exchange. They have three primary functions: ATP generation, Cabstances . When mibstances . Even unbstances .In the skeletal muscle of COPD patients, decreases in number of mitochondria per unit area and in biogenesis are frequently observed . ReducedChanges in mitochondrial content and apparent changes in muscle fiber types are the main manifestations of mitochondrial adaptation to external stimuli. The entire adaptation process is called mitochondrial biogenesis . Peroxisi.e., ATP synthase) utilizes the released energy to catalyze the oxidative phosphorylation of adenosine diphosphate (ADP) and produce ATP [Mitochondrial respiration is the most efficient energy metabolism mechanism in skeletal muscle. It depends on the MRC, which consists of complexes I-IV and is located on the inner mitochondrial membrane. When electrons transfer through the MRC, the MRC enzyme complex V , and lipids [Reactive oxygen species (ROS) are involved in various types of signal transduction in cells. Low levels of ROS can play a protective role as an essential cellular signal . Howeverd lipids . Mitochod lipids -44.OS in COPD skeletal muscle comes from exogenous and endogenous pathways. Exogenous OS comes from harmful substances, such as free radicals and carbon monoxide in smoke. Endogenous OS is derived from the self-defense response of inflammatory cells and proliferation of ROS . MitochoThe mitochondrial dysfunction of COPD skeletal muscle is caused by a combination of factors, predominantly related to smoke exposure, hypoxia, and insufficient physical activity.Harmful exposure factors such as smoking are the primary causes of COPD. Smoking severely damages the normal physiological structure of the lungs, causes breathing difficulties, and induces a series of toxicological damages to skeletal muscles via local and systemic effects . Studies2) [Oxygen is vital to maintenance of normal skeletal-muscle mitochondrial function. Hypoxia is a common predisposing factor for SMD in patients with COPD, mediating damage to mitochondrial structure and function. Sauleda et al. found that in hypoxemia, the activity of complex IV, as a terminal enzyme of the MRC, is altered in COPD patients and is inversely related to arterial oxygen partial pressure (PaO2) . This fi2) . Hypoxia2) . In addi2) .Physical inactivity is prevalent in patients with COPD and pronounced during exacerbations of the disease . PhysicaExercise, a core PT for SMD in COPD patients, can ameliorate mitochondrial dysfunction by improving mitochondrial phenotype and redox status.Different exercise strategies offer different degrees of mitochondrial regulation. One study found that eccentric exercise in patients with COPD can significantly improve muscle strength and mass, but no considerable change in mitochondrial function was observed. Meanwhile, concentric exercise can significantly change mitochondrial biogenesis and MRF . A possiStudies have also found that despite no significant change in skeletal-muscle mitochondrial density after exercise in patients with COPD, single-joint exercise in these patients can elevate skeletal-muscle maximal oxygen uptake and muscle oxygen supply more than whole-body exercise . This reDifferent exercise styles have similar effects on mitochondrial improvement. Endurance exercise has long been the preferred method for pulmonary rehabilitation and has the characteristics of repetition, duration, and low intensity. Maltais et al. demonstrated that endurance exercise, specifically 12 weeks of cycling training, reduces exercise-induced lactic acidosis and improves the oxidative capacity of skeletal muscle in patients with moderate to severe COPD . In addiThe effects of different exercise intensities on mitochondrial dysfunction are also similar. High-intensity interval training is effective in PT for patients with COPD . One stuIn conclusion, exercise’s ability to improve mitochondrial dysfunction in COPD skeletal muscle cannot be ignored. The current evidence shows that the effects of different exercises on mitochondrial dysfunction in skeletal muscle in COPD patients are not entirely consistent. Both concentric and single-joint exercise can better promote mitochondrial respiratory improvement. Eccentric exercise maximizes skeletal-muscle function, both endurance exercise and resistance exercise can better restore the mitochondrial redox state of COPD skeletal muscle, and high-intensity interval training may be better at improving mitochondrial dysfunction in these patients.Taking the above into account, exercise rehabilitation programs for COPD patients should be formulated based on patient cardiopulmonary function, disease severity, and maximization of benefits. For COPD patients with poor exercise tolerance and impaired cardiorespiratory function who are in the initial stage of enhanced physical activity, we recommend eccentric exercise and single-joint exercise that exchange low cardiorespiratory load and low metabolic cost for muscle strength and quality. COPD patients with relatively good exercise ability can choose concentric, endurance, and resistance exercises that enhance mitochondrial function better. Exercise intensity should be based on the patient’s exercise tolerance. No studies have yet definitively identified the best exercise for COPD patients. As discussed above, the optimal exercise strategy combines different exercise modalities, enhancing patient muscle function and potential effectors, for example, the combination of concentric and eccentric exercises or of endurance and resistance exercises. The relevant literature on the effect of exercise on mitochondrial dysfunction in skeletal muscle is shown in Normal mitochondrial density and biogenesis are essential for energy metabolism in skeletal muscle. Long-term regular exercise can increase mitochondrial number and biogenesis, resulting in good adaptation of muscle cells . This reThe effect of exercise on COPD skeletal-muscle mitochondrial biogenesis is closely related to activation of PGC-1α-related signaling pathways. PGC-1α inhibits the ubiquitin proteolytic system and myostatin to reduce muscle protein degradation and is a signaling node for exercise-triggered mitochondrial density and biogenesis . Among tMitochondria are where the energy required for COPD skeletal-muscle movement is generated, and normal MRF is a necessary condition for skeletal-muscle exercise capacity. During exercise, the MRF of skeletal muscle is upregulated. ADP and mitochondrial substrate concentrations are significantly increased during exercise compared with the resting state, thereby promoting generation of ATP to continuously supply energy . StudiesNumerous studies demonstrate that exercise can improve mitochondrial dysfunction in COPD skeletal muscle by upregulating MRF. After a 7-week rehabilitation process combining endurance and strength exercises, Calvert et al. found reduced blood ammonia levels and lactate accumulation and loss of adenine nucleotides in COPD patients, which increased ATP synthesis and fully supplied the body’s energy metabolism . This fiThe normal work of mitochondrial respiration depends on the MRC. Mitochondrial complex I is the largest protein complex on the inner mitochondrial membrane and is responsible for 95% of mitochondrial respiration . PreviouThe redox reaction is the electron transfer process between oxidants and antioxidants. As important carriers of the redox reaction, mitochondria go through a series of redox states, reflecting the level of cellular metabolism . Therefo2O2 production in skeletal muscle compared with the resting state [2O2 production is slow during muscle contraction, which might be due to enhanced catalase activity [i.e., enhanced antioxidant scavenging. Current research finds that the enhanced ROS-scavenging effect of the body’s antioxidants is related to nuclear factor erythroid 2-related factor 2 (Nrf2). When the body is stimulated by excessive ROS, the upstream protein Kelch-like epichlorohydrin (ECH)-associated protein 1 (Keap1), which regulates cellular localization and Nrf2 content, is inhibited. The bound Nrf2 is released and transferred to the nucleus, where it interacts with antioxidant response element (ARE). The combination of Nrf2 and ARE increases the activity of antioxidant enzymes, neutralizes excess ROS, and regulates redox state balance [Under constant stimulation of ROS production both externally and internally, the body’s antioxidant defense system plays an important role in inhibiting ROS. Antioxidant defense systems are divided into two categories: enzymatic antioxidants, such as SOD, catalase, and glutathione peroxidase (GSH-Px); and nonenzymatic antioxidants, such as vitamins C and E . An expeng state . Consistactivity , i.e., e balance , 95. Tol balance . These rTo the best of our knowledge, exercise might enhance the mitochondrial-antioxidant defense mechanism of COPD skeletal muscle to inhibit ROS generation and lower OS by regulating mitochondrial-electron leakage through UCPs. Studies show that exercise can restore UCP3 content in the muscles of COPD patients, alter ROS generation, improve oxidative capacity, and protect mitochondria from the toxic effects of OS . HoweverInterestingly, in recent years, more studies have focused on whether the underlying mechanisms by which different exercises affect mitochondrial metabolism are similar . As demoThe occurrence of SMD in COPD is universal, adjustable, and related to the degree of respiratory symptoms. It can easily lead to various adverse consequences such as falls and fractures. Among the many factors contributing to skeletal-muscle structure and function impairment in COPD, mitochondrial dysfunction is at the center of the pathogenic mechanism network, directly affecting muscle energy metabolism. From the perspective of mito-chondrial damage, various studies have found that it influences muscle strength and endurance, which nicely explains the internal relationships between molecular mechanisms, especially in the presence of OS. Smoke exposure, hypoxia, and insufficient physical activity are important risk factors affecting mitochondrial function. The interaction of multiple factors ultimately changes mitochondrial density and biogenesis, decreases MRC function, and increases mitochondrial ROS production. However, the underlying mechanism linking mito-chondrial dysfunction with impaired structure and function in COPD skeletal muscle is not fully understood. Understanding mitochondrial dysfunction in COPD skeletal muscle is a critical premise and challenge in exploring the COPD therapeutic principle and effect, which will help identify a precise target in future studies.To the best of our knowledge, this article is the first to delve into the underlying mechanisms by which exercise improves mitochondrial dysfunction in COPD skeletal muscle. Notably, targeting mitochondrial dysfunction in COPD skeletal muscle through exercise training is prospective. Exercise is a non-drug treatment that effectively promotes an increase in mitochondrial density and biogenesis, improves MRF, and regulates redox status, meaning that it can reverse mitochondrial dysfunction and prevent further loss of skeletal muscle. We also recommend appropriate exercise options for COPD patients with different disease severities and exercise abilities, which can provide a reference for clinical application. Based on the existing research, the combination of different exercise strategies such as endurance and resistance exercises seem to improve various functional indicators of mitochondria, thereby restoring the optimal state of COPD skeletal muscle. However, relatively few studies focus on exercise in terms of mitochondrial phenotype and the molecular mechanism of changes in COPD skeletal muscle. No study has comprehensively compared the improvement effects of different exercise times, intensities, and forms, nor has any particular study focused simultaneously on the three aspects of mitochondrial-phenotype changes improved by exercise. Therefore, the effects of different exercise patterns, durations, and intensities on mitochondrial damage should be further studied. In future work, we will focus on mitochondria to explore the exact mechanism by which exercise improves treatment effect in COPD patients, providing new ideas for exercise rehabilitation programs.


Non-invasive brain stimulation (NIBS) has emerged as one of the methods implemented in stroke rehabilitation. Cerebellar stimulation has gained research interest as an alternative strategy to cortical stimulation, based on the role of the cerebellum and corticocerebellar tracts in different motor and cognitive functions. This review investigates the role of the cerebellum in motor and cognitive rehabilitation following cerebral stroke using NIBS techniques combined with other therapies . Fifteen randomized clinical trials were included. The majority of the literature findings point towards the cerebellum as a promising neurostimulation target following stroke of the cerebral cortex. Findings concern mostly rehabilitation of gait and balance, where cathodal transcranial direct current stimulation (tDCS) and intermittent theta-burst stimulation (iTBS) of the contralesional cerebellar hemisphere produce, in the presented clinical sample, improved performance and plasticity changes in the corticocerebellar network, combined with other rehabilitation methods. Data regarding aphasia rehabilitation are scarce, with right cerebellar tDCS exercising some impact in individual linguistic functions combined with language therapy. Based on recent data concerning cerebellar functions and corticocerebellar networks, along with the development of clinical protocols regarding non-invasive cerebellar (NICS) application, the cerebellum can prove a crucial intervention target in rehabilitation following stroke. Despite ongoing dispute on the definition of stroke, the term has been classically referred and applied to the WHO definition 1970) of ‘rapidly developed clinical signs of focal  disturbance of cerebral function, lasting more than 24 h or leading to death, with no apparent cause other than of vascular origin’ 970 of ‘r. To thisTMS has the potential of modulating the excitability of specific brain areas using a coil that produces a rapidly alternating magnetic field, which in turn induces an electric field in the target-area. The latter is defined either by specific anatomical landmarks, or by identifying the motor ‘hot-spot’, the region where the highest motor-evoked potential is generated . When cuTranscranial magnetic stimulation can be applied using different protocols depending on the outcome of interest, either as a single pulse with a current intensity sufficient to produce action potentials, or as repeated stimulation (rTMS) with low (<1 Hz) or high (>5 Hz) frequency. Theta-burst stimulation (TBS) involves applying trains of 50 Hz pulses in a continuous (cTBS) or intermittent manner (iTBS). As a rule, low-frequency rTMS and cTBS cause a temporary suppression of excitation (inhibition), as indicated by decreased motor evoked potentials (MEP) amplitudes, while high-frequency rTMS and iTBS facilitate excitation, as indicated by increased MEP amplitudes .tDCS creates a potential difference between two or more applied electrodes and alters the neuronal membrane potential inside the formed electrical field. Depending on the current direction  and axonal orientation, neuronal depolarization or hyperpolarization occurs, with a general rule of increase in excitability with anodal tDCS, and decrease in excitability with cathodal tDCS . tDCS haAn ischemic lesion can cause important changes in the complex neural networks of the lesioned tissue. The most important finding is the remapping observed in the perilesional cortex . A cruciThanks to spontaneous neuroplasticity, the central nervous system (CNS) compensates in a varying degree for the functional disorder cause by stroke. Despite that, spontaneous reorganization may cause maladaptive plasticity, or prove insufficient for the retrieval of lost functionality . The samIn many cases, the motor deficits arising from a supratentorial stroke are associated with the phenomenon of crossed cerebellar diaschisis (CCD), which includes reduced blood perfusion and loss of spontaneous Purkinje cell firing . CCD conThe cerebellum plays a crucial role in various motor and non-motor functions, and especially regulating motor and non-motor behavior through adaptation and constant update of internal models, which facilitate error-based learning and motor control. Synaptic plasticity through LTD and LTP plays an important role in this process, by regulating Purkinje cell-parallel fiber synapses firing and consequently the cerebellar signaling to the cerebral cortex . In thisVarious studies have been published regarding the functional influence of cerebellar transcranial magnetic stimulation or direct current stimulation on motor cortex. Modulation of cerebellocortical connectivity depends on the different protocols used, as well as the interpersonal differences concerning circuitry and neurotransmission. A systematic review by Tremblay et al. (2016) attempts to collect experimental findings regarding NICS (non-invasive cerebellar stimulation) effect on different neurophysiological components of motor cortex neuroplasticity in healthy subjects and neurological patients, by using low- and high-frequency rTMS, iTBS or cTBS, tDCS, and PAS (paired-associative stimulation). The main study findings are the decreased CBI following cerebellar rTMS/tDCS, and the neuroplastic changes on M1 following PAS .The field of cerebellar neuromodulation is rapidly evolving in the 21st century, both on an experimental and therapeutic level, in consistence with the advanced knowledge concerning cerebellar function in health and disease. The cerebellum has become a crucial neuromodulation target due to the high density of neural cells, the electric properties it possesses, and its involvement in various motor, cognitive, and affective loops . So far,For this review, a systematic search of the literature was conducted in the databases PubMed and Scopus from July to October 2022 to include studies published between 2010 and 2022, by using the following keywords independently or combined by advanced search strategy: cerebellar stimulation, cerebellar TMS, cerebellar tDCS, stroke recovery .Studies that met the following criteria were considered for inclusion in the present review: (a) they were classified as randomized clinical trials (RCTs); (b) participants suffered from cerebral stroke; (c) NIBS were under investigation; (d) both pre and post intervention data were presented. Studies were excluded according to the following criteria: (a) other study designs ; (b) study protocols; (c) conference abstracts; (d) studies performed in animals; € reports not published in English; (f) retracted papers; (g) articles assessing different interventions (not NIBS) as well as (h) studies that also included participants with other neurological conditions ; (i) studies with participants without stroke or with cerebellar stroke; (j) studies without a control/placebo group or with healthy control group. Retrieved abstracts were meticulously assessed. In case of inability to establish if a study met the inclusion criteria, we reviewed the full text.The following data were extracted according to standardized data extraction forms, and qualitatively analyzed: author, year of publication, number of participants, TBI stage, interventions assessed, cognitive status, cognitive domains assessed, study design, outcomes measures, duration and frequency of the intervention, results. Two independent reviewers (Ε.-A.Ν. and G.N.) conducted the literature search and data extraction independently. Discrepancies were resolved by a third author (A.Ν.).The extensive literature search provided 6681 results , which after the removal of duplicates were limited to 2370. Following title assessment, 1838 studies were excluded, while following further evaluation with abstract assessment 434 studies were not considered relevant for inclusion in this review. A total of 98 full texts were analyzed, and ultimately 28 clinical studies specifically concerning the use of NICS in stroke rehabilitation were assessed for eligibility for inclusion in this review. Seven studies were excluded based on the site of lesion (cerebellar stroke), two studies were case reports, and in four studies, full text or abstract was unavailable. Consequently, 15 randomized clinical trials (RCTs) were included for analysis. A total of 8 studies concerned the use of TMS protocols, in particular rTMS, iTBS, and PAS, and 7 studies concerned the use of tDCS. In total, 13 studies investigated aspects of motor recovery following cerebral stroke, and 2 studies investigated language recovery. Below are presented the findings of the RCTs based on the stimulation method applied and the functional target of recovery. The flowchart of the literature search is presented in Applications regarding cerebellar TMS in cerebral stroke rehabilitation according to current literature concern dysphagia, gait-balance, and limb spasticity as outcomes of interest. Both rTMS and TBS have been studied but there is a prominent application of TBS, the main reason being that it can induce long-lasting cortical excitability with both lower stimulation intensity and training duration .Initial findings concerning cerebellar TMS efficacy in rehabilitation of dysphagia arose from a study by Jayasekeran et al. (2011), where the interaction between the cerebellum and cortical activity in the pharyngeal representation area was investigated using TMS in healthy subjects, with an enhancement of pharyngeal motor evoked potentials (PMEPs) and increase in amplitude observed following paired pulse cerebellar TMS . FurtherThe efficacy of an rTMS protocol was investigated by Zhong et al. (2021) in 143 patients with subacute stroke and certified dysphagia, with an important as well as similar improvement of dysphagia recorded in 2- and 4-weeks following intervention in all intervention groups . The stuBalance is considered an aspect of postural adaptation, and cerebellar hemispheres play an important part in motor adaptation. Improved balance is associated with improved gait performance, reduced risk of falls, and overall improved functional independency following stroke .In the study of Koch et al. (2018), 36 chronic stroke patients with hemiparesis and balance disorder were submitted in daily sessions of either contralesional cerebellar iTBS, or sham iTBS supplemented with physiotherapy. Study findings included an overall increase in the Berg Balance Scale (BBS) score, which was clinically presented with a transition from supported to independent gait with reduced risk of falls, as well as a narrowing of stepping amplitude reflecting a steadier gait in participants who received iTBS, as well as enhanced neural activity in the posterior parietal cortex of the lesioned hemisphere . In the Spasticity, a velocity-dependent elevation of muscle tone, is one of the most common debilitating findings in stroke survivors. Considering the role cerebellum exerts over motor control, along with positive findings from the use of cerebellar TBS in motor disorders such as levodopa-induced dyskinesia and cervical dystonia, cerebellar TBS has emerged as a promising experimental protocol for spasticity alleviation.In a study by Dawei Li et al. (2021), three different TMS protocols were compared regarding their effects in muscle spasticity and limb dyskinesia in 90 chronic stroke patients see  who alsoCerebellar tDCS has been studied in post-stroke gait-balance disorders and aphasia. In a randomized clinical trial by Zandvliet et al. (2018), 15 patients with chronic supratentorial stroke and standing balance disorder (<56 score in BBS), and 10 healthy age-matched controls were submitted in an anodal cerebellar tDCS protocol during a postural tracking task. Standing balance performance was evaluated in three static positions, with open or closed eyes, and in a tandem position, with an important decrease in center of pressure (CoP) parameters recorded in the more demanding tandem position in patients who received contralesional anodal stimulation . Ranjan Based on the positive findings of a previous study concerning the enhanced efficacy of robotic therapy in chronic stroke patients after application of cerebral tDCS combined with transcutaneous spinal direct current stimulation (tsDCS), Picelli et al. (2018) conducted a new study of a similar protocol, applying this time cerebellar tDCS in 20 chronic stroke patients. The theoretical framework lies on the neuronal basis of gait generation and control, which consists of complex control mechanisms including supraspinal structures and spinal centers that form the central pattern generators, cerebellum being an important node of this network . The autThe potential uses of non-invasive cerebellar stimulation in aphasia rehabilitation have emerged through findings concerning cerebellar involvement in non-motor aspects of language, such as phonetic and semantic fluency . IncreasThe present review is the first one to focus on the role of the cerebellum in motor and cognitive rehabilitation after cerebral stroke using NIBS techniques combined with other therapies . Findings arising from published RCTs raise a promising perspective regarding the use of non-invasive cerebellar stimulation following cerebral stroke. In the motor domain, improvement in performance is recorded in patients suffering from dysphagia, gait, or balance disorders and limb spasticity/dyskinesia. Language recovery findings are only preliminary with recorded improvement in verb naming and retrieval. Most studies are consistent in showing improvement in the clinical question, with differences in improvement and efficacy degree rising from protocol and stimulation techniques variability, as well as interindividual differences regarding stroke severity or mechanisms of recovery. Gains in performance are recorded in all studies following a combined intervention of non-invasive stimulation and physical/speech therapy or newer rehabilitative techniques such as robotic therapy.Regarding the functional influence of non-invasive cerebellar stimulation on motor cortex, in general it has been shown to induce plasticity mechanisms at the cortical level and consequently at the cerebellocortical networks, demonstrated both by enhanced connectivity, and by modulation of MEP’s amplitudes and CBI . Part ofThe concept of cerebellar stimulation engages, according to the current literature, both the interhemispheric inhibition model and the vicariation model for stroke plasticity. For example, cathodal stimulation of the contralesional cerebellar hemisphere improves gait performance compared to anodal ipsilesional cortical stimulation in the study by Picelli et al. (2018), by indirectly enhancing the cerebellothalamocortical pathway and reversing CBI, but the same protocol did not show a predominance compared with cathodal ipsilesional cerebellar stimulation, which in turn enhances the activity of the contralesional cerebral hemisphere, in the following study .The parameters of the cerebellar s”Imu’ation were defined mostly based on data from previous studies, which employed MRI reconstruction and neuronavigation techniques. Regarding tDCS, the electrodes were applied in the cerebellum according to the international 10/20 or 10/5 EEG systems, with current intensity either at 1.5 mA or 2 mA. In the studies concerning TMS, a figure-of-eight coil was applied tangentially to the skull in a position defined by previous studies as optimal for eliciting MEPs of the highest amplitude in the cortical region of interest. For example, in studies investigating dysphagia recovery, the coil was positioned 4.3 cm lateral and 2.4 cm below the inion, an area corresponding to the pharyngeal cortical representation area , while iCathodal tDCS of the contralesional cerebellar hemisphere and iTBS of the contralesional cerebellar hemisphere show the most frequent application in published studies concerning gait/balance rehabilitation, with recorded efficacy related to the facilitation of the lesioned cerebral hemisphere. A comparison of efficacy of applied protocols concerning limb spasticity or dysphagia cannot be made based on included studies, as there is protocol variability, but in both cases, TMS protocols have been applied and proven efficient. It is confirmed that motor output concerns the function of a multimodal network of structures, with stimulation of a network node affecting further network areas . ReorganFindings concerning the use of non-invasive cerebellar stimulation in aphasia rehabilitation following stroke remain limited, and arise from limited-population studies, with tDCS proving a more favorable technique. There is no obvious superiority between anodal/cathodal stimulation, possibly due to the complexity of the cerebellar cortical folding which may cause simultaneous depolarization and repolarization of neurons, and lead to different overall effects, while subjective anatomical and neurophysiological parameters play an important part in different polarity effects . MarangoPlasticity mechanisms in the corticocerebellar system following cerebral stroke, and findings regarding cerebellar involvement in gait and balance control, language, and cognitive networks, render the cerebellum an important neuromodulation target. Different study and stimulation protocols, small clinical samples, and subjective differences in circuitry and stroke semiology prevent the extraction of safe conclusions regarding the use of NICS in stroke rehabilitation, but study findings open encouraging perspectives for establishing its use. Factors affecting its efficacy, but also setting its possible advantage over cortical stimulation, include the location and size of stroke lesion affecting current transmission, the white matter tracts structural integrity, the degree of disorder, the residual functionality, the time passed from stroke appearance, and the location, protocol, and duration of stimulation .The main limitations of this review are the small number of clinical studies published so far, as well as the small clinical sample included. Furthermore, the variability of NICS techniques and the non-established consensus on the investigated protocols render the interpretation of findings and the comparison between protocols challenging. Most studies lacked an individualized approach concerning the side of stimulation, while stimulus or current intensity varied between studies. In addition, some studies lacked long-term follow-up so lasting effects of the stimulation cannot be yet observed. Moreover, not all studies included neurophysiological measurements that could interpret clinical and functional outcomes and could provide prognostic values, while in the studies that included neurophysiological measurements, the results did not reflect the clinical outcome. Current results regarding NICS in stroke recovery have a proof-of-concept value based on published studies. Although a direct comparison between cerebellar and cortical stimulation cannot be made in the context of stroke recovery based on current literature, it is made clear that cerebellar stimulation can provide therapeutic alternatives that can prove useful depending on the functional target, the lesion site/severity, and the individual mechanisms affecting plasticity.The main challenge of cerebellar stimulation lies in the question of its future response to the perspectives it has established according to current findings. Future studies are expected to include a larger population sample for better reproducibility and credibility of various findings. In addition, further customization of NICS protocols regarding stroke rehabilitation will enable better comparison between studies. This might be assisted by the development of mathematical models that will predict current diffusion and distribution and will increase stimulation precision , as wellDespite encouraging results, long-term effects of cerebellar stimulation in sustaining functional gain need to be recorded and evaluated considering other factors affecting rehabilitation. Moreover, policies that will assist the transfer of arising findings from experimental to wide clinical practice need to be implemented, taking into consideration the patients’ benefit.Overall, establishment of stroke as a main short- and long-term functional disability source renders necessary the application of new rehabilitation protocols that will respond to its broad semiology and varying functional impact. The cerebellum can prove a crucial intervention target in rehabilitation following stroke. Non-invasive cerebellar stimulation appears to be a promising tool in this context, and its use in clinical practice remains to be established in the direct future.


The functionalization of nanomaterials with suitable capping ligands or bioactive agents is an interesting strategy in designing nanosystems with suitable applicability and biocompatibility; the physicochemical and biological properties of these nanomaterials can be highly improved for biomedical applications. In this context, numerous explorations have been conducted in the functionalization of silver (Ag) and gold (Au) nanomaterials using suitable functional groups or agents to design nanosystems with unique physicochemical properties such as excellent biosensing capabilities, biocompatibility, targeting features, and multifunctionality for biomedical purposes. Future studies should be undertaken for designing novel functionalization tactics to improve the properties of Au- and Ag-based nanosystems and reduce their toxicity. The possible release of cytotoxic radicals or ions, the internalization of nanomaterials, the alteration of cellular signaling pathways, the translocation of these nanomaterials across the cell membranes into mitochondria, DNA damages, and the damage of cell membranes are the main causes of their toxicity, which ought to be comprehensively explored. In this study, recent advancements in diagnostic and therapeutic applications of functionalized Au and Ag nanomaterials are deliberated, focusing on important challenges and future directions. The functionalization of nanomaterials with suitable capping ligands or bioactive agents is an interesting strategy in designing nanosystems with suitable applicability and biocompatibility; the physicochemical and biological properties of these nanomaterials can be highly improved for biomedical applications ,2,3,4. ISurface-functionalized Au NPs with distinctive optical, electrical, photothermal, and specific surface plasmon resonance features have been explored as strong colorimetric and fluorescent sensors that can provide immense opportunities for broad biomedical applications . DiffereThe visualization of Au nanomaterials can simply be performed by photometry and electron microscopy due to the high electron density, specific absorption, and scattering in the visible and near-infrared region . These n−1 and correlation coefficient = 0.993); the limit of detection was ~1.74 μg mL−1 with a limit of quantification of ~5.30 μg mL−1 [Citrate-functionalized Ag NPs were prepared for the sensitive detection of glutathione in human biological blood and serum samples using surface-enhanced infrared absorption spectroscopy . Accordi μg mL−1 . In anot μg mL−1 .−2) for ~10 min, offering a smart platform for advanced theranostics in future bio- and nanomedicine [Biocompatible glycol chitosan–coated Au NPs were designed as photoacoustic contrast agents for the cellular imaging of cancers . The premedicine .Au NPs with a large surface area can be deployed as effective platforms for therapeutic agents, allowing for the efficient binding of targeting agents and drugs. In one study, Au nanomaterials stabilized by pullulan were coupled with 5-fluorouracil and folic acid for targeted drug delivery and cancer imaging ,51. ThesRNA-conjugated Au NPs were fabricated to efficiently knock down the formation of luciferase in cancerous cell lines (HeLa cells) transfected luciferase. These conjugates exhibited a longer lifetime (~6 times longer) than the double-stranded RNA; in addition, the cells could simply penetrate with no need of deploying any transfection agents . MoreoveCurrently, numerous in vitro and in vivo studies have been performed on cancer diagnosis and therapy applications of Au NPs due to their unique physicochemical properties and high versatility ,55. FuncA novel nanosystem was constructed via the conjugation of gadolinium (Gd)(III) complexes and prostate-specific-membrane-antigen–targeting ligands to the surfaces of Au NPs for targeted magnetic resonance–guided radiotherapy with improved targeting precision and efficacy. The designed nanocontrast agents exhibited robust magnetic resonance contrast and radiation therapy efficiency (in vitro and in vivo), providing encouraging nanosystems for cancer therapy and diagnosis with good tumor accumulation and high in vivo radiation dose amplification. However, toxicity and biocompatibility issues along with their clinical translation ought to be systematically addressed . SimilarWith their controlled treatment area of light exposure, photothermal (PTT) and photodynamic therapy (PDT) have been broadly applied in bio- and nanomedicine. The performance of PDT includes the excitation of a photosensitizer at a precise wavelength. The photosensitizer transfers its energy to the molecular oxygen and thus produces reactive oxygen species (ROS), destroying tumor cells through necrotic or apoptotic cell death pathways. PTT can be applied as a therapeutic method in which a therapeutic factor is stimulated with a specific wavelength of light and energy to create heat through photo absorption, thereby killing the surrounding cancer cells. In this method, a near-infrared laser (NIR) is applied to irradiate the tumor cells locally in order to uniformly enhance the temperature in the tumor and inhibit damage to healthy cells; effective ablation occurs when the center of the tumor reaches a therapeutic temperature above 55 °C ,65.Au NRs and nanoshells have been widely used in PTT because of their robust absorption in the NIR region . Since AAu NRs were modified with PEG toward the synthesis of PEG-Au NRs and conjugated by a TPS at the end of the PEG chain, thus creating TPS-PEG-Au NRs. Energy absorption under NIR-laser irradiation enhanced the temperature of the Au NRs and formed ROS derived from talaporfin sodium. The combination of nanoplatforms and NIR-laser irradiation showed outstanding cytotoxic effects against lung cancer cells under an NIR laser. Au NSs are effective multifunctional NPs simultaneously used in PTT and PDT using NIR-laser irradiation. The outcomes offer an advantageous model of nanomedicine for cancer therapy using NIR lasers . Kayani The functionalization of nanomaterials with aptamers can offer a highly efficient diagnostic assay to probe target cells and tissues. Thus, various studies have recognized aptamers for imaging, detection, and intracellular analysis . An apta−1 Ag NTs and an 800 nm laser, although the viability of the cells pre-incubated with gold nanorods and subjected to NIR stimulation was only reduced by 20% [−1 under irradiation at 800 nm laser [Lavandula angustifolia Mill extracts  and (11-mercaptoundecyl)-N,N,N-trimethylammonium cation  to produce nanosystems with dual functions including antimicrobial and proangiogenic performances for wound healing in diabetic mice [S. aureus. They showed robust bactericidal activities (in vivo) and enhanced the generation of collagen fibers and epithelialization, offering favorable materials for wound healing and treating chronic wound infections [In addition, PEG-Ag NPs were synthesized with enhanced bactericidal effects toward l effect . Impresstic mice . Consequfections .Au NPs have been established as promising nanomaterials for bone regeneration. These NPs can improve the osteogenic differentiation of mesenchymal stem cells, act as osteogenic agents, increase the grafting of bone implants, and hasten bone formation in bone defects ,79. ResuEpigallocatechin gallate (EGCG) is the most prevalent polyphenol in green tea and can effectually scavenge ROS and can reduce autoimmune arthritis by moderating Th17/Treg. EGCG can inhibit osteoporosis in an in vivo model and display a therapeutic effect on periodontitis in rats . EGCG-AuDespite the various advantages of surface-functionalized Au- and Ag-based nanomaterials, the use of these NPs systems for tissue targeting in the human body or in vivo has not been more generally studied relative to other nanomaterials, such as lipid-based systems . ConsideAnother challenge is finding the mechanisms and specific roles of functionalized Au and Ag NPs in tissue engineering . These aFunctionalized nanomaterials have been widely explored in the fields of drug and gene delivery, cancer theranostics, imaging and diagnosis, and anti-infections. Among the nanomaterials, functionalized Au/Ag-based nanosystems possess the special benefits of improved biocompatibility, good biodegradability, targeting, specificity and selectivity, low toxicity, high drug loading capacity, and sustained and controlled drug release behavior. Surface functionalization of these nano-agents can help to overcome concerns about biosafety, thus providing excellent opportunities for developing smart nanosystems with gene and drug delivery, bioimaging and biosensing, and cancer theranostics applicability. However, more elaborative studies should still be conducted to fill the existing research gaps, especially pertaining to nanotoxicological assessments, optimization processes, and related mechanisms.The selection of suitable capping agents with multifunctional features, as well as surface modification techniques, can play important roles in the development of these Ag- and Au-based nanosystems. Notably, the majority of studies in this field are focused on the laboratory scale; thus, future explorations should be moved toward the large-scale production and functionalization of these nanomaterials utilizing appropriate capping agents. Toxicity and biosafety assessments are vital in acquiring multi-functionalized nanosystems with high efficiency and low or negligible adverse side effects and should be comprehensively performed; this is particularly true for clinical translational studies.


Precision oncology continues to challenge the “one-size-fits-all” dogma. Under the precision oncology banner, cancer patients are screened for molecular tumor alterations that predict treatment response, ideally leading to optimal treatments. Functional assays that directly evaluate treatment efficacy on the patient’s cells offer an alternative and complementary tool to improve the accuracy of precision oncology. Unfortunately, traditional Petri dish-based assays overlook much tumor complexity, limiting their potential as predictive functional biomarkers. Here, we review past applications of microfluidic systems for precision medicine and discuss the present and potential future role of functional microfluidic assays as treatment predictors. Precision oncology is important for patient treatment. Here the authors review the current applications of microfluidic systems to cancer precision medicine, and discuss the issues that must be addressed prior to getting these technologies into the clinic. Precision oncology is based on the use of agents that specifically target the molecular alterations exhibited by the patient’s tumor cells, as opposed to the traditional “blockbuster” chemotherapy agents targeting basic cell functions . Precision medicine originated in the early 2000s, when advances in genome sequencing made it possible for clinicians and researchers the analysis of the tumor’s DNA to identify driver mutations . Enabled by this information, clinicians could target the patient-specific molecular alterations that were fueling tumor growth. This approach led to the emergence of multiple compounds targeting the tumor’s molecular alterations  that are currently used to treat numerous malignancies such as leukemia , breast cancer , and melanoma 6, the field of molecular analysis has evolved to include additional analysis techniques such as RNA sequencing, proteomics, or metabolomics. The rationale behind these approaches was to improve the accuracy of outcome predictions8. These genomic-based approaches to precision oncology have had real but limited success7. Numerous factors are limiting the success of precision medicine, including technological challenges, limitations regarding information integration, and biological problems9. Thus, functional assays that directly evaluate treatment response on live cells and consider additional factors such as the tissue of origin, the tumor microenvironment, or the immune system, may offer a compelling complement11. More specifically, functional assays are defined as tests that quantify the performance or behavior of cells to directly evaluate treatment response, and broadly include all cell-based assays that use non-genomic technologiesAlthough precision medicine has traditionally relied on the genomic analysis to identify targetable mutations in tumor cellsOverall, microfluidic technologies offer a versatile toolbox to address current challenges regarding tumor sample collection and analysis, as well as improve the predictive power of functional assays for precision medicine applications. Thus, in this perspective, we discuss the current and rising opportunities of microfluidic systems for precision medicine.9. Functional assays are defined as tests that quantify the performance or behavior of cells to directly evaluate treatment response, and broadly include all cell-based assays that use non-genomic technologies12. The setups for these assays can be relatively simple, including patient-derived cells cultured as a 2D monolayer; or highly complex such as 3D hydrogels with stromal, immune, and tumor cells culture on transwell inserts that also include endothelial and lymphatic monolayers. Thus, functional assays aim to describe an evolving system where the effect of molecular changes , as well as microenvironmental factors , can be captured over time. Arguably, since functional assays can evaluate critical features of the tumor microenvironment , these assays could provide more robust predictors to identify the optimal treatment11. Further, complex functional assays also allow for the observation of emerging properties resulting from the interaction of multiple cell types , and microenvironmental components . Overall, functional assays provide an interesting approach to monitor tumor evolution and treatment response.As the lively debate about the predictive power of multi-omic technologies continues, functional assays may play a pivotal role13. Researchers argued that the main limiting factor of functional assays based on 2D Petri dishes was their capacity to capture the tumor complexity. Thus, the emergence of microfluidic systems, is a promising alternative. In recent years, the terminology around microfluidic systems has become more nebulous with the emergence of concepts like organ-on-a-chip and microphysiological systems. Whereas microfluidic systems is an umbrella term encompassing all microfluidic devices regardless of their application for precision medicine, including sample isolation, or analyte detection; organ-on-a-chip and microphysiological systems both refer to 2D/3D modeling constructs commonly used to predict tissue/tumor behavior and treatment response. In this context, organ-on-a-chip has traditionally focused on recapitulating organ functions such as the breathing of the lung. In contrast, microphysiological system does not necessarily require microfluidic technology and aims to recapitulate physiological functions. Here, we discuss the contributions and potential of microfluidic-based technologies to precision medicine, including devices focused on sample processing, analyte detection, and, especially, functional response.Fostered by the promise of more robust predictions, researchers explored the use of functional assays decades ago, pharmacologically treating patient-derived cells in 2D Petri dishes to predict patient response but these assays showed very limited predictive capacity14.Although these models have been used to study multiple aspects of human disease , their potential for cancer research has experienced the fastest growth in the last decade, and researchers have started to explore their use in the clinic for functional diagnostics and precision medicine17. Microfluidic platforms are an attractive alternative due to their great potential to mimic complex tissue architectures involving multiple biological structures such as vasculature, stromal barriers, nutrient gradients, or hypoxia19. Microfluidic models commonly rely on culturing one or multiple cell types  in microfluidic devices that recapitulate the structure and environment of the tissue of interest. Most of the advantages offered by microfluidic models derive from the predictive fluid dynamics at the microscale . Regarding the second challenge, microfluidic models may have a better chance to predict treatment response compared with traditional assays given their potential to mimic the complex tumor microenvironment. Tumors are complex 3D structures where multiple environmental factors include nutrients, waste products, chemokines, cell types , and structures  are present. All of these microenvironmental factors potentially affect treatment response and they become instrumental for treatments targeting stromal components such as anti-angiogenic or immunotherapyBox 1 Advantages of microfluidic modelsGeneration of tailored in vitro cell culture systems. Microfluidic models are well-suited to generate biologically-relevant 3D geometries such as tubular structures, perfusable vascular networks, or patterned co-cultures. Mimicking in vivo topography changes cell behavior, leading to results that can closely resemble in vivo observations.Capacity to control microenvironmental factors. Microfluidic systems excel at controlling biochemical gradients , mechanical forces, and spatial configurations that modulate tumor progression. Examples include collagen fiber alignment, hypoxia, and nutrient gradients, and chemotactic gradients.Coupling with detection and analysis platforms. Sample processing and analysis techniques can be directly integrated with microfluidic devices . Likewise, the highly controllable thickness and geometry of the platforms can facilitate optical inspection and microscopy, making them compatible with high-content image analysis systems.38. A seminal example by Saadi et al. reported using parallel liquid streams to study the chemotactic effects of an EGF gradient on breast cancer MDA-MB-231 cells. Another relevant example was provided by Shimizu et al., who leveraged a microfluidic gradient generator to study the effects of histamine on endothelial cells in the context of allergy-associated vasodilation/vasoconstriction. Further advances in microfabrication led to sophisticated models that included numerous gradient-generation units, making these platforms amenable for high-throughput screenings39, which are instrumental in assessing multiple therapeutic options in a timely manner.Cancer cells rely on multiple signaling molecules such as growth factors or cytokines to modulate their environment and promote tumor growth. Microfluidic devices can generate a highly predictable flow pattern, providing more control over molecule diffusion, a known limitation of traditional assays like the Transwell platform, and presenting an opportunity for early applications of microfluidics in cell biology. Some of these approaches included leveraging molecular diffusion across two parallel streams to create a gradient perpendicular to the flow direction or generating a tree-like network of connected microfluidic channels to create stable non-linear polynomial gradients. The gradients’ profile is controlled by modifying the channel design and dimensions and the pressure applied at the inlets. These gradient generators have been leveraged to explore the chemotactic properties of numerous compounds  in a variety of cells 40) to study the effect of breathing motions on lung cancer proliferation, dormancy, and response to therapeutic agents41. Other studies have investigated the effects of mechanosensing in TME models using microfluidics. Another example of the mechanical cue is the effect of collagen density, which correlates with a worse prognosis in breast cancer. A study investigating the effects of increased collagen density using microfluidics reported an increased secretion of IL-6 (a pro-inflammatory cytokine) that compromised the barrier function of lymphatic vessel models in the presence of MDA-MB-231 cells42. Despite the many examples investigating the effect of mechanical cues using microfluidics45, their application to cancer remains relatively unexplored, and creates an opportunity for more studies in this field.A major recent focus in cancer research is the effect of mechanical cues and 3D architecture on cell behavior, such as particular matrix architecture and stiffness, tissue deformation, and fluid flows. These forces modulate cell function, such as proliferation and migration. Therefore, integrating these cues may dramatically affect cell response to therapeutic agents. An example of studies leveraging microfluidics to study the effects of mechanical cues was reported by Hassell et al. They leveraged the lung-on-a-chip model  as physical barriers that would later be lined with an endothelial monolayer. Endothelial monolayer models mimicked the blood vessel endothelial wall, expressed relevant cell–cell adhesion molecules , and formed a barrier that modulated molecule diffusion . In a particular study, HT-1080 fibrosarcoma cells were embedded in the hydrogel, and their intravasation through the confluent endothelial monolayer was monitored. The authors showed that RAW264.7 macrophages increased the intravasation rate in a TNF-α-dependent manner, highlighting the role of stromal cells during cancer progression. However, this model exclusively relied on the use of an endothelial monolayer, whereas in vivo blood vessels are highly dynamic, having the capacity to remodel their architecture and form new vessels depending on the nutrient and oxygen demand. More advanced models have focused on capturing in vitro angiogenesis and vasculogenesis, which imply the formation of new blood vessels from pre-existing vasculature and during embryogenic development respectively. Kim et al. used a similar microdevice that included five parallel microchannels to embed endothelial cells  in a hydrogel solution in the central microchannel 49. Although most models have traditionally focused on blood vessels, other studies have also evaluated the capacity of these platforms to mimic lymphangiogenesis, i.e., the formation of new lymphatic vessels50, showing differences in permeability and protein secretion between endothelial and lymphatic vessels51. Overall, these results demonstrated the capacity of microfluidic models to recapitulate critical features of the tumor vasculature during tumor development.Other studies perfused cancer cells through the blood vessel to study tumor extravasation and metastasis, demonstrating the distinct interactions of different cancer cells with nearby vasculature. Another field of study has been demonstrating the potential of various stromal cells to affect the vasculature 2), thereby imposing a continuous gradient across the culture chamber52. Following a similar approach, other studies investigated cell response to nutrient  gradients, demonstrating the effect of glucose gradients in cell migration and proliferation53. The main advantage of this approach is the capacity of the user to control the gradient profile, although they require specialized equipment to control the environment. Conversely, other models have leveraged cell metabolism to generate oxygen and nutrient gradients55. Nutrients and oxygen flowed through only the flanks of a central chamber, and as cells consumed oxygen and nutrients, they generated a gradient across the chamber. As a response to these gradients, cancer cells transitioned from a proliferative state to a dormant phenotype . These platforms also allowed cell retrieval from different spatial locations  for downstream analysis and sub-culture to study cell stress response and resilience55. Many other microfluidic platforms have aimed to mimic the cellular composition and organization of the tumor microenvironment by co-culturing cancer cells with relevant stromal cells . Recent examples included a breast cancer model reported to mimic the transition from ductal carcinoma in situ (DCIS)56  to malignant invasive ductal carcinoma. In this model  on tumor growthOverall, microfluidic technologies have reached enough maturity to mimic virtually any geometrical structure found in biology, including complex and highly structured examples such as the human eye or neuronal networks, and we believe these models are ready for the clinic from a material engineering Fig. . AlthougMicrofluidic devices designed for the clinic broadly fall into two different applications: (1) analyte detection for molecular diagnostics, and (2) disease modeling for functional diagnostics. The former application focuses on detecting, and even in some cases quantifying, the presence of a specific analyte  to diagnose or inform clinical decisions. The second application includes those microfluidic systems designed to analyze functional responses on live cells, which hold clinical potential but are not yet established.Molecular diagnostics commonly rely on detecting or quantifying the expression of a given mutation or protein . Microfluidic systems offer superior capacity for analyte detection compared with traditional systems  given their smaller volume and the predictable fluid behavior. Thus, researchers have explored a diverse body of applications for these technologies for cancer diagnosis. Most of these systems include microfluidic channels with a specific geometry designed to separate the different analytes  from blood cells) present in the sample . Next, the purified analyte is collected for downstream analyses, or in some cases analyzed in situ within the microfluidic devices. Researchers have used microfluidic devices to analyze a large variety of analytes from human samples including CTCs, proteins, circulating tumor DNA (ctDNA), and exosomes.9 cells. Thus, CTCs provide valuable insight into tumor behavior and treatment response, but they are extremely challenging to analyze with traditional methods. Microfluidic devices leverage the fluid properties at the microscale such as lateral deterministic displacement, inertial focusing, or Dean vortex flow61 and antigen labeling 63 to isolate CTCs, offering superior performance compared with their macroscopic counterparts64. Devices like the CTC-iChip leverage multiple of these principles to isolate CTCs in a label-free manner from whole blood, whereas their compatibility with mass manufacturing techniques  ensures they can be rapidly produced and distributed through the healthcare system65.Solid tumors commonly shed cells that travel through the bloodstream as individual cells or small cell clusters (<100 cells). However, their relative abundance compared with other blood cells like T cells is extremely low, commonly ranging from 1 to 10 cells per 1 mL of whole blood, which typically contains >1066, which holds potential for patient profiling and stratification. Although there is a correlation between tumor size and levels of ctDNA, traditional technologies have achieved moderate success translating ctDNA analysis into an actionable treatment plan. The main limitation regarding ctDNA analysis interpretation is that normal cells also secrete DNA into the bloodstream, known as cell-free DNA (cfDNA), making discrimination between normal and tumor cfDNA extremely difficult67. Thus, the main challenge is identifying tumor mutations in ctDNA in a saturated pool of wild-type cfDNA 68. Therefore, microfluidic systems designed for digital PCR  present a promising alternative. These systems encapsulate individual ctDNA and cfDNA molecules in aqueous droplets to perform a PCR reaction within each droplet. The use of a fluorescent probe dissolved within each droplet allows a computer to detect the presence of extremely rare mutations in complex DNA mixtures, which can be as low as one mutated molecule every 200,000 wild-type molecules. Recent pilot studies involving a small number of patients have used digital PCR to diagnose different tumor subtypes that are difficult to stratify , identifying the best therapeutic option and significantly improving patient outcomes.Similar to CTCs, primary and metastatic tumors also secrete ctDNA into the bloodstream69. Microfluidic devices for protein detection in clinical samples are one of the more mature applications, leading to multiple commercially available products in the market70. These platforms are capable of successfully detecting proteins in the femtomolar range using as little as a few nL of blood or plasma by implementing a digital ELISA test, which is based on a similar concept to digital PCR but encapsulating individual proteins instead of nucleic acids in aqueous droplets73. Finally, recent studies have started to use microfluidic devices for cancer-associated exosome detection and characterization. Exosomes are small (70–120 nm) lipidic vesicles derived from multivesicular bodies that contain a heterogenous cargo like nucleic acids, proteins, lipids, or metabolites, and modulate multiple aspects of tumor biology such as tumorigenesis, metastasis, and drug resistance75. Given their complex cargo and higher stability, they may offer a powerful alternative for cancer diagnosis compared with the other analytes described77. Microfluidic devices for exosome analysis in the clinic commonly rely on immune-affinity methods such as the use of antibodies targeting exosome-specific antigens)79 although some recent reports are showing the potential of size-based techniques81. There have been a few examples of microdevices for exosome isolation, like the herringbone chip, used in a clinical setting82. These devices can process several mL of whole blood outperforming traditional exosome isolation techniques such as ultracentrifugation or magnetic beads.Other circulating biological materials may offer additional valuable insight. Tumors commonly secrete abnormal levels of proteins into the bloodstream, including hormones, cytokines, and growth factorsOverall, microfluidic devices for molecular analysis have shown significant progress in the last decade, and, arguably, they are ready to offer a competitive alternative to other traditional techniques, especially in the CTC field Fig. . However83. Thus, as microtechnologies continue to evolve, the use of these models is already being explored in the clinic. A notable example was a microfluidic device that successfully predicted multiple myeloma patients’ response to bortezomib in a model comprising patient-derived tumor cells and autologous stromal cells 84. The results showed that stromal cells were necessary to correctly stratify responders and non-responders, highlighting stromal cells’ relevance in drug response. Despite the small cohort assessed in this study , the results underscored the potential of microfluidic models to inform the clinical decisions for hematological cancers using functional readouts. Functional microscale assays are currently in the spotlight to assess solid tumor cytotoxicity. A recent study presented a microfluidic assay to evaluate the metastatic potential of breast cancer specimens85, revealing a specific phenotype and genotype of highly migratory cells, consistent with metastatic capacity in animal models. This study helped pinpoint specific molecular alterations such as RAS or PI3K pathway mutations associated with enhanced migration and metastatic potential targeted with clinically tested compounds to assess the therapeutic effects on cell migration. This model could help stratify patients according to the metastatic potential of their tumors to define an effective treatment plan. Other microfluidic platforms have been used to evaluate the role of patient-specific stroma on cancer invasion86. A noteworthy example was the study by Truong et al., in which breast cancer cells were co-cultured with patient-derived normal or cancer-associated fibroblasts. The microdevice allowed the authors to identify a new targetable gene  associated with stroma-induced tumor invasion86.As previously described, microfluidic models and organ-on-a-chip platforms are, arguably, better equipped to mimic the tissue complexity required to provide a predictive response in some cancers. Thus, functional microfluidic assays allow multiple readouts to be collected from a small sample, which increases the statistical power of the assay and potentially mitigates the N-of-1 problem90. An example is head and neck cancer, which is highly heterogenous and no reliable predictors of tumor metastasis exist. A recent study isolated fibroblasts from different head and neck cancer patients and co-cultured them with a biomimetic lymphatic vessel in a microfluidic device  despite aggressive treatments combining brain surgery, radiation, and chemotherapy94. In a recent study, the authors used decellularized porcine brain tissue to generate an off-the-shelf bio-ink92. Next, the authors isolated glioblastoma and endothelial cells from multiple patients and combined the with the bio-ink. Using a 3D bioprinter, they generated a circular culture containing two concentric rings, the endothelial cells were seeded in the outer ring whereas cancer cells were confined to the inner layer. The synthetic tissue was encapsulated within a microfluidic device to create an oxygen gradient across the tissue. The results demonstrated that the model mimicked in vivo tissue organization, showing cancer stem cells, invasive cancer cells, and endothelial cells distributed in different spatial locations. Finally, the authors treated the model with concurrent chemoradiation, which involves radiotherapy combined with chemotherapy (temozolomide), and demonstrated that the model predicted the in vivo clinical response.Some microfluidic platforms also leverage 3D bioprinting and decellularized scaffolds to create highly organized structures that mimic complex tissue organizations Fig. 92. Gliob95. Most chemotherapy agents target proliferating cells, potentially affecting other normal cells such as bone marrow precursors. As a consequence, clinicians commonly have to modify patient treatment due to bone marrow damage. To address this problem, Chu et al used a microfluidic model to capture the complex environment observed in the bone marrow and study drug toxicity and pathophysiology in a patient-specific manner96. This model included two chambers horizontally separated by a porous membrane. The authors seeded patient-derived bone marrow precursors and stem cells in a 3D hydrogel in the upper chamber, whereas they seeded endothelial cells in the lower chamber to mimic the vasculature. After two weeks in culture, the bone marrow precursors differentiated into multiple blood cell progenitors such as neutrophil, megakaryocyte, and erythroid progenitors. The authors used the model to evaluate bone marrow toxicity of several drug candidates including 5-FU or AZD2811 and evaluated cellular damage on the multiple blood cell progenitors.Other authors are adopting an alternative approach to precision medicine by leveraging microfluidic devices to rapidly identify drugs that will lead to severe off-target and deleterious side-effectsOne of the traditional limitations of functional assays is that they offer limited throughput compared with molecular testing, where numerous molecular targets  can be analyzed simultaneously. Therefore, researchers are also leveraging microfluidic devices to generate high-throughput systems to streamline the screening process of dozens or even hundreds of drug candidates Fig. . To over26, and few have moved beyond small patient cohorts, limiting the clinical relevance of their results99. Overall, we believe that more and larger studies are still necessary, but microfluidic models are already moving toward precision medicine and functional diagnostics104.Overall, accumulated evidence suggests that microfluidic models could provide a robust tool for precision medicine. On the other hand, most of these models remain inside research labsMicrofluidic models are becoming robust and mature enough for use in clinical scenarios from a technological perspective. However, there are still a few challenges that should be addressed. Here we discuss current and future challenges that must be overcome to make the next generation of functional precision oncology a reality.105. However, PDMS presents critical limitations, including low production volume or absorption of small hydrophobic compounds . Alternative materials  circumvent these limitations and are now being used both in academia and industry. Another challenge that should be addressed is microdevice operational complexity, which remains high compared with other techniques performed in the clinic 26. To address the issue of consistency, pathology labs and manufacturing companies have developed standardized guidelines and protocols that make immunohistochemistry or PCR analyses more approachable and universal across different laboratories, hospitals, and institutions. A similar effort will be required to enable microscale models to be widely adopted. Microfluidic device manufacturers should consider the current workflows and infrastructure in clinical labs and the industry to ensure successful translation into the clinic.Despite the rapid progress exhibited in the field in the last few years, several technical challenges should be addressed to integrate this technology into the healthcare system. First, most microfluidic devices have been fabricated in polydimethylsiloxane (PDMS), which is well suited for prototyping new devices107. Tumors are spatially heterogeneous structures that can show different genetic, protein, and functional  profiles depending on the specific tissue fragment analyzed106. Depending on the tissue fragment used to generate the model, the treatment response may change. Although there is no definitive solution to this problem, the small number of cells required for functional microfluidic assays could help mitigate this issue. Microfluidics enables creating of multiple models from different biopsy sites, providing a more comprehensive picture of the tumor phenotype. Additionally, new clinical technologies, such as in vivo molecular imaging, are being developed to evaluate intratumor heterogeneity with minimally-invasive techniques111. These techniques rely on labeled probes that recognize molecular alterations in human cells including radio-labeled HER2 antibodies, or PSMA detection, allowing radiologists to generate a molecular map of the tumor heterogeneity in real time114. Despite sharing the limitations of other molecular approaches, molecular imaging could be particularly useful to evaluate tumor heterogeneity, pinpointing the optimal biopsy locations for downstream functional microfluidic assays.Intratumor heterogeneity is a significant challenge in precision oncology114. Microfluidics could be instrumental in solving this problem by using precision fluid flows to mechanically disrupt tissue. Reported prototypes also require small volumes, which improves the speed and efficiency of tissue digestion, therefore preserving cell viability and minimizing sample loss117. Additionally, since only a small number of cells are needed, in vitro expansion may not be required, helping retain in vivo phenotypes.Multiple studies have shown that sample processing via chemical/mechanical digestion, cell isolation, and culture expansion lead to numerous alterations including loss of molecular markers, decrease in cell viability, or disruption of the tumor microenvironment. This alteration of the molecular/functional profile can bias the results obtained with patient-derived samples. To address this problem, the NIH has allocated specific funding to develop more effective and robust protocols and techniques to work with patient-derived cancer samples118. iPSCs can be obtained from the patient skin and then differentiated in vitro into the other cell types required, reducing the need for additional, and sometimes impossible to get, patient-derived samples119. Future advances in iPSC technology may help expand cancer and tissue types that can be modeled using functional approaches.One important consideration before building patient-specific functional organotypic models is the number of tissues or organs that should be included. There is no doubt that several cell types  and organs play a role in tumor drug response. Due to the critical role that the immune system plays in tumor evolution and the remarkable recent results shown by immunotherapies, the inclusion of an immune component in functional assays becomes key. However, obtaining biopsies from all the different organs and tissues required for this approach is generally impossible. The use of induced pluripotent stem cells (iPSC) cells provides an appealing alternative to solve this problemThe ultimate goal of functional microfluidic models is to improve patient outcomes. To achieve this goal, we envision future studies to identify and validate clinically relevant models and readouts. Drug response would be analyzed in different configurations of the functional model and compared with the patient response, therefore identifying the most predictive configuration and readouts Fig. . The spe120. There is a clear need to reduce unnecessary costs for these new precision oncology therapies. Other cost-cutting initiatives already proposed for clinical-grade diagnostic assays include simplification of model setup and operation, which would reduce the need for specialized personnel89. Therefore, it will be imperative that clinicians and biologists evaluate the minimum model complexity needed to predict patient response26. Although much remains to be done in this field, we believe the current high cost of these assays will diminish as technology advances. We also expect that during the validation phase, these functional models may in some cases identify new molecular biomarkers  that may suffice for a subset of patients and result in effective patient stratification guidelines. Microfluidic functional assays may present additional challenges compared with molecular tests from a regulatory point of view. Most molecular tests rely on the detection of specific markers ), which makes the development of guidelines and experimental protocols more straightforward compared with functional assays. In microfluidic functional assays, the experimental conditions  may have a critical impact on the response observed, thus increasing the need for standardized protocols and guidelines. Interestingly, recent successes in cell-based immunotherapies have demonstrated that functional approaches can be applied in the clinic (https://www.fda.gov/BiologicsBloodVaccines/CellularGeneTherapyProducts/ApprovedProducts/default.htm). The manufacturing of genetically modified immune cells is also a very complex protocol and standardized protocols are essential to guarantee product efficiency and safety. The Food and Drug Administration (FDA), the European Medicine Agency, and other similar institutions are developing guidelines to implement these cell-based immunotherapies in the healthcare system implement functional assays in the healthcare system. We believe regulatory organizations could develop similar guidelines for functional microfluidic assays based on recent and soon-to-come preclinical and clinical data.Many clinical-grade diagnostic tests are initially developed to address either a new clinical need or to identify patients for treatment with a new therapeutic agent . This paradigm of integrated drug development-companion diagnostics has become mainstream and has improved patient care by both identifying patients most likely to benefit from a given therapy and minimizing the risks of overtreatment. Optimizing treatment via trial-and-error often comes with avoidable toxicity side effects while cancer keeps progressing. Microfluidic models that can more accurately reflect the benefit of these new treatments have great potential in eliminating treatment trial-and-error but do not yet meet the CAP (College of American Pathologists)/CLIA  guidelines. These certifications are often expensive processes requiring demonstration of analytical validity followed by clinical validation in prospective trials. These costs add up to an already oversized and constantly increasing investment in healthcare As the complexity and adaptability of cancer continue to be unveiled, it is becoming increasingly clear that a genomic-centric approach to precision oncology may not suffice to meet the clinical challenges and clinical opportunities presented with recent therapeutic advances. Propitiously, recent technological advances are enabling a more integrative precision oncology paradigm. This situation presents an opportunity for functional microfluidic assays, which offer the potential to go beyond a single snapshot in time to capture the complex behavior of cancer adaptation and subsequent response to treatment. We anticipate that functional microfluidic assays will complement -omics-based disciplines to propel the successful implementation of precision oncology for the next generation of cancer care.