Spatial metabolomics offers an opportunity to demonstrate the drug-resistant tumor profile with metabolic heterogeneity, and also poses a data-mining challenge to reveal meaningful insights from high-dimensional spatial information. In this review, we discuss the latest progress, with the focus on currently available bulk, single-cell and spatial metabolomics technologies and their successful applications in pre-clinical and translational studies on cancer drug resistance. We provide a summary of metabolic mechanisms underlying cancer drug resistance from different aspects; these include the Warburg effect, altered amino acid/lipid/drug metabolism, generation of drug-resistant cancer stem cells, and immunosuppressive metabolism. Furthermore, we propose solutions describing how to overcome cancer drug resistance; these include early detection during cancer initiation, monitoring of clinical drug response, novel anticancer drug and target metabolism, immunotherapy, and the emergence of spatial metabolomics. We conclude by describing the perspectives on how spatial omics approaches  could be further developed to improve the management of drug resistance in cancer patients.Resistance to drug treatment is a critical barrier in cancer therapy. There is an unmet need to explore cancer hallmarks that can be targeted to overcome this resistance for therapeutic gain. Over time, metabolic reprogramming has been recognised as one hallmark that can be used to prevent therapeutic resistance. With the advent of metabolomics, targeting metabolic alterations in cancer cells and host patients represents an emerging therapeutic strategy for overcoming cancer drug resistance. Driven by technological and methodological advances in mass spectrometry imaging, spatial metabolomics involves the profiling of all the metabolites (metabolomics) so that the spatial information is captured  Cancer drug resistance occurs when the tumor is getting insensitive to drug treatment, which also explains tumor recurrence and metastasis . ResistaThe complexity of mechanisms underlying cancer drug resistance requires omics-driven systems approaches. Our focus has long been on mapping the alterations at the genome, epigenome and proteome levels, even though changes at the metabolome level are more closely related to the drug resistance phenotype . Howeverbona fide  have been characterised  technologies, holds great promise for an improved understanding of cancer drug resistance. As its name suggests, spatial metabolomics can globally profile metabolites, lipids, drugs, and other small molecules; profiled so in the spatial context of cells, tissues, organs, and even the whole organism . It is wona fide . Via spacterised , and metcterised . Moreovecterised . Recentlcterised .In the remaining sections of this review, we provide an in-depth appraisal of the technologies and methods currently available for bulk, single-cell, and/or spatial metabolomics, and discuss how these latest advances have improved our understanding of mechanisms underlying the metabolic reprogramming in tumor responses to anticancer drugs. We will also discuss the outstanding challenges involved and share our perspectives on further developing integrative spatial omics approaches to maximise the potential of metabolomics in dissecting cancer drug resistance.Metabolites are essential components of the complex biological system, which reflect the environment in which the cells are located. They are tightly correlated with the effects of drugs, the nutritional status of cells, and the influence of other external factors. The term “metabolomics” describes the global identification and quantification of metabolites. Technologies used to identify and quantify metabolites globally have evolved from classic bulk metabolomics, single-cell metabolomics and spatial metabolomics, and to the emerging spatial single-cell metabolomics.in vitro cultured cells, tissues, and biofluids  , matrix-in situ within a sample from different biological tissues and organs, using MSI techniques, thus optimising and complementing traditional metabolomics approaches. The histology of cancer tissues is highly dynamic and complex  or desorption electrospray ionisation-MSI (DESI-MSI). The imaging process involves the virtual separation of the sample into many “pixels”, each described by a mass-to-charge (m/z) spectrum . Specialin situ metabolomics on intact tissue sections, thus allowing for comprehensive analyses of metabolic heterogeneity  can confer a chemo-resistant phenotype.GLUT1/3/4) widely studied in cancer. A study using lung and breast cancer models revealed a potent anti-tumor effect of WZB117, a GLUT1 inhibitor, that could decrease glycolysis and reduce intracellular adenosine triphosphate (ATP) levels (GLUT4 (GLUT3 (The entry of glucose into the cells is facilitated by a transporter family known as the glutamines (GLUTs) , with th) levels . In anots  to regulate the heat shock response . HSF1 caThe role of amino acids has been gaining popularity in the field of cancer metabolism in recent years. Cancer cells reprogram the amino acid metabolism to maintain tumor progression and support the complex microenvironment in driving the resistance to anticancer therapies. Amino acid provides resistant cells with drug-specific adaptations, drive the epigenetic modulation of drug-resistant cancer cells, modulate the tumor microenvironment to anticancer therapies, mediate tumor immune evasion, and regulate CSCs to promote cancer aggressiveness.Amino acid metabolism provides resistant cancer cells with specific adaptive mechanisms to counteract anticancer drugs Figure 4Amino acid metabolism drives the alteration of epigenetics, which supports the survival of drug-resistant cancer cells Figure 4Amino acids play an important part in the complex crosstalk of the tumor microenvironment and the regulation of tumor-induced immunosuppression, thus leading to cancer drug resistance. Cancer cells have a specific mechanism to educate the neighboring stromal cells to adapt their metabolism. In pancreatic ductal adenocarcinoma, cancer cells were reported to promote pyrimidine synthesis by the tumor-associated macrophages (TAMs), leading to an increase in the production of deoxycytidine by macrophages, which directly competes with gemcitabine and hinders its efficiency as a treatment drug for cancer cells . In addihigh/CD24low CSCs population in TNBC cell lines, leading to the inhibition of methionine adenosyltransferase 2A, which is responsible for SAM biosynthesis in CSCs  to prevent chemotherapy toxicity in leukemia stem cells . Early s in CSCs . Likewis in CSCs . These fDe novo lipid biosynthesis drives tumor resistance. Compared to normal cells, cancer cells tend to increase lipogenesis rather than depending on dietary lipids or using lipids synthesised from liver cells. Aberrant de novo fatty acid biosynthesis provides a continuous supply of resources for membrane synthesis, energy consumption, and signaling to cancer cells. Fatty acid biosynthesis depends on a series of enzymes, including but not limited to: ATP-citrate lyase (ACLY), Acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN). ACLY was reported to be upregulated in BRAF-mutation melanoma and promoted mitochondrial biogenesis and OXPHOS to support tumor growth and resistance to vemurafenib  to reduce the activation of prodrugs; the other is the conversion of the drugs into intermediate metabolites for macromolecule synthesis  and drug transporters (such as P-glycoprotein) have been reported to be overexpressed in mitoxantrone-resistant cells; they act to remove chemotherapeutic agents , represents new forms of cancer therapy. Immune functions are related to tumor cell metabolism; for example, gain-of-function mutations in isocitrate dehydrogenase (IDH) resulted in the production of D-2-hydroxyglutarate, an oncometabolite that altered T-cell metabolism to impair CD8+ T cytotoxicity and interferon-gamma signaling in patients with IDH1 mutant gliomas . Down-reChanges in the metabolome are most closely related to the drug-resistant phenotype . Five meChemoresistant cancers tend to display an aggressive clinical outcome and early recurrence. During drug treatment, a small subpopulation of cancer cells can remove the anticancer drug, and thus the developing resistant cells become the dominant population. Screening the subset of chemoresistant cancer cells at an early stage might prove effective in inhibiting cancer drug resistance. Metabolomics is a powerful tool that can unbiasedly identify cancer biomarkers that drive cancer drug resistance.Metabolites are stable in the serum; hence, it is possible to establish a noninvasive method for the early detection of the metabolic biomarkers of cancer. Conventional methods for the detection of pancreatic cancer include the estimation of the serum level of the carbohydrate antigen 19-9 . The potCirculating tumor DNA (ctDNA) tests can be used for early detection, including non-invasive dynamic detection of cancer and monitoring of clonal evolution . It can Routine approaches to assess drug resistance during chemotherapy treatment have been used; for example, seven metabolites  have been identified to be associated with chemoresponses . In one in situ. Resected NSCLC tissue specimens obtained after neoadjuvant chemotherapy were subjected to high-resolution mass spectrometry, and the data generated was used to develop an approach for evaluating the response to neoadjuvant chemotherapy in patients with NSCLC  alkylating agents that target the DNA, causing either single-strand breaks or crosslinking of DNA to prevent the cell from proliferating; (ii) antimetabolites that disrupt DNA synthesis and cell division by inhibiting the formation of normal nucleotides or by direct interaction with DNA, thereby preventing the extension of DNA strands; and (iii) enzyme inhibitors that affect DNA replication and the cell cycle. As a folic analog, the antimetabolite (5-fluorouracil) is a chemotherapy drug that inhibits thymidylate synthase and decreases the thymidine triphosphate level for DNA replication. These traditional chemotherapy drugs are non-specific to tumors and may inhibit the proliferation of normal cells, causing severe side-effects, such as drug resistance. Targeting cancer metabolism pathways involved in tumor development and metastasis is increasingly becoming feasible for cancer therapy. Metabolic reprogramming is one of the hallmarks of cancer cells; altered metabolic pathways  support the rapid proliferation of cells. Furthermore, these dysregulated metabolic features are also linked to therapeutic resistance in cancers .Metabolites in cancer are linked to the activation of proto-oncogenes and the inactivation of tumor suppressor genes. Upstream regulators of metabolic pathways  are important targets for anticancer drugs. Targeting HIF prevents the metabolic shift or adaptation of tumor cells to hypoxia. Anticancer agents (such as PX-478) that reduce the HIF-α level have demonstrated potent anti-tumor effects . Rapamycin vitro activities against cancers . Pyruvate kinase catalyses the conversion of phosphoenolpyruvate and adenosine diphosphate into pyruvate and ATP, respectively; it was reported to be associated with tumor growth and cisplatin resistance  have demonstrated anticancer effects by reducing the uptake of glucose and slowing down glycolysis rate . These asistance .ex vivo .Spatial metabolomics can be carried out to overcome drug resistance. Early detection to identify the metabolic changes in the tumors during the preliminary stage via histology-guided MALDI-MSI was performed based on the metabolites, and rare IDH-mutated NSCLC was screened by evaluating the levels of the oncometabolites  can be applied to unveil the tissue complexity, heterogeneity, intracellular signaling, and disease progress underlying the drug resistance. For example, the concordance between gene expression and staining of the corresponding proteins can be assessed in the same tissue regions to validate the findings of spatial transcriptomics , 171. FuMetabolic reprogramming has been recognised as a hallmark of cancer in promoting therapy resistance and many others. Targeting metabolic alterations in cancer cells and host patients represents an emerging therapeutic strategy for overcoming cancer drug resistance, particularly at the advent of spatial metabolomics. This review describes the latest progress in technologies and methods currently available for  metabolomics, and discusses how these latest advances have improved our understanding of the metabolic mechanisms underlying tumor responses to anticancer drugs, along with the potential of using metabolomics to overcome drug resistance and the perspective on further developing integrative spatial omics approaches to dissect cancer drug resistance.More specifically, we have discussed the specific metabolic programs and adaptations that exist in drug-resistant tumors, how these adaptations depend on both the drug and the origin of the tumor, and how they contribute to drug resistance. Accumulated evidence strongly suggests that combining many first-line chemotherapeutic agents with metabolic drugs holds great promise in increasing the drug efficacy. Moreover, a better understanding of the altered metabolism in different drug-resistant cancers and the distribution of metabolites and the tumor microenvironment through spatial metabolomics is essential to further improve the outcomes of cancer therapy. This additional information will provide insights into the molecular mechanisms of resistance, which will help in identifying novel metabolic targets that can be used for combined treatment. Finally, this knowledge may be applied to identify the prognostic biomarkers for drug response, which could drive current therapies by predicting the drug response based on the metabolic state of the tumor, thereby contributing to more effective personalised medicine.ZZ and HF: Conceptualisation. ZZ, CB and HF: Data curation and writing - original draft preparation. ZZ, CB and HF: Visualisation and interpretation. LJ, SW, KW and CL: Writing - reviewing and editing. HF: Supervision. All authors contributed to the article and approved the finalised version.


We carried out a scoping review to characterize the primary quantitative evidence addressing changes in key individual/structural determinants of substance use risks and health outcomes over the first two waves of the COVID-19 pandemic in the United States (US). We systematically queried the LitCovid database for US-only studies without date restrictions (up to 6 August 2021). We extracted quantitative data from articles addressing changes in: (a) illicit substance use frequency/contexts/behaviors, (b) illicit drug market dynamics, (c) access to treatment and harm reduction services, and (d) illicit substance use-related health outcomes/harms. The majority of 37 selected articles were conducted within metropolitan locations and leveraged historical timeseries medical records data. Limited available evidence supported changes in frequency/behaviors/contexts of substance use. Few studies point to increases in fentanyl and reductions in heroin availability. Policy-driven interventions to lower drug use treatment thresholds conferred increased access within localized settings but did not seem to significantly prevent broader disruptions nationwide. Substance use-related emergency medical services’ presentations and fatal overdose data showed a worsening situation. Improved study designs/data sources, backed by enhanced routine monitoring of illicit substance use trends, are needed to characterize substance use-related risks and inform effective responses during public health emergencies. In the United States (US), the confluence of the coronavirus disease 2019 (COVID-19) pandemic with the opioid overdose epidemic, which began over 20 years ago, has been appropriately termed a “clash of epidemics” ,4.In the years leading up to the pandemic, the opioid crisis had been compounded by the growth in polydrug use and the illicit supply of fentanyl and other highly potent synthetic opioids ,6. IncreWhereas previous review articles have examined various interactions between the COVID-19 pandemic and substance use in the US ,15,16,17Based on guidance from Munn et al. and Peters ,22, we sWe developed a search strategy with the broadest substance use-related key terms  without Relevant study descriptions and main findings were systematically extracted . No formOur search strategy returned a total of 2440 articles. After the title/abstract screening and full-text review processes, 37 articles were included based on our criteria .As detailed in A fifth (n = 9) of the reviewed studies provided evidence pertaining to changes in illicit substance use frequency, contexts, and associated risk behaviors. For this domain, we make a distinction between studies that explicitly sampled people with a history of illicit substance use or substance use disorders (SUD) and those that sampled people without any prerequisites regarding their history of substance use.Studies that reported on similar quantitative outcomes are presented in Two cross-sectional studies recruited participants with an established history of substance use or SUD. Jacka et al.  looked aAmong populations for whom recruitment was not related to history of substance use or SUD, we observed mixed trends in illicit substance use during the pandemic. Janulis et al.  presentePalamar and Acosta  reportedIn contrast, a study by Duncan et al.  comparedTwo studies provided insight from toxicology data. Young et al.  describeNiles et al.  also fouThere is scant evidence regarding changes in interpersonal aspects or contexts of substance use. Mistler et al.  found a p < 0.01).Among sexual minority men, Starks et al.  showed tOnly four articles addressed changes in illicit drug market dynamics, with available quantitative results detailed in Palamar and Acosta’  survey oAnother study by Palamar et al.  presenteWith 16 out of 37 articles providing data related to changes in treatment and harm reduction services access, the majority utilized patients’ medical records retrospectively, either from MOUD clinics or state and national prescription databases, to ascertain desired outcomes.p < 0.001). Contrastingly, other studies also indicate reduced access or capacities for MOUD programs in some settings despite the regulatory changes. This includes Herring et al.’s [In recognition of pandemic conditions, the Substance Abuse and Mental Health Services Administration (SAMHSA) issued regulatory changes in March 2020 around the delivery of MOUD, including increases in the number of take-home doses allowed as well as lifting administrative limits placed on the number of new patients for whom providers can issue prescriptions . The effet al.’s  report oet al.’s  surveyedet al.’s  reportedet al.’s  report fThe review also found several studies reporting the degree of success in the continuation of treatment services by healthcare providers for people with SUD. Among a sample of individuals with jail discharges, Duncan et al.  found anEvidence on changes in the delivery of harm reduction services following the onset of the pandemic was limited, although multiple studies indicated that there were disruptions in access to these services. Jones et al.  analysedWith a total of 15 studies identified with outcomes related to this domain, most evidence addressing this question reported outcomes related to fatal or non-fatal overdose from opioids.p < 0.001), then gradually decreased to 0.7% by the last week of December 2020.Five studies presented findings on non-fatal overdose outcomes from data at the national level. Holland et al.  presenteData from 18 emergency departments (ED) in 18 different US states  would app < 0.001). However, as with the study by Pines et al. [An additional five studies presented findings on non-fatal overdose outcomes using region- or facility-specific data . Following national trends, in Louisville, Kentucky, Shreffler et al.  showed ts et al. , the sams et al. . Data frs et al.  also fous et al. . The sams et al. . RelativOne study, Ridout et al. , provideFive studies presented findings on fatal substance-related overdoses. Shreffler et al.  analyzedOur study provides a characterization of evidence regarding the impacts of the COVID-19 pandemic on a comprehensive range of outcomes concerning illicit substance use within US-specific contexts over the first two waves. Other scoping reviews that shared similar goals have either presented generalized global findings  or were Prevailing evidence presented in this review poses challenges in evaluating the exact magnitude or direction of changes in illicit substance use frequency and behaviors. Except for two studies utilizing biological samples to determine substance use before and post-pandemic ,33, all Although evidence on changes in health services access for those with illicit substance use are more robust compared with the previous domains, the outcomes reported are, once again, varied and fragmented across different study settings. In general, the reduction of limits on MOUD prescribing practices by SAMHSA gave rise to enhanced and lower-barrier access primarily within individual MOUD clinics in terms of buprenorphine initiation, prescription, and dosage duration ,38,39,40The strongest body of evidence found in this review pertains to changes in substance use-related health outcomes, specifically for overdose outcomes. Among the 10 studies investigating changes in EMS utilization for substance use, ED/EMS overdose data were presented differently, with some focusing on changes in terms of the absolute number and others focusing on the relative proportion of substance use-related encounters. Despite our efforts to reconcile the information through calculating common measures, transparent interpretation proves challenging. Whereas most of the studies showed increasing trends in relative terms, only a subset found absolute increases in the encounters related to substance use ,53,54,55The search strategy and scope of the review in the present study relies on PubMed through LitCovid, which omits any articles that are not indexed in PubMed or other adjacent literature databases. Due to the inclusion criteria specified, the current study also potentially omits any articles presenting evidence on relevant changes in substance use-related outcomes during the time period of interest but without any explicit reference to COVID-19. Likewise, it would be challenging to determine the influence of COVID-19 on the findings of studies not explicitly carried out within the perspective of the pandemic.In terms of the data presented, we noted the schism of data quality between studies with self-reported measures and process-based measures such as ED/EMS time series data, drug tests results, and drug seizures data, with potential issues of self-reporting bias in the case of the former that can undermine the validity of findings from the respective studies. Similarly, differing interpretations of ED/EMS data across studies in relative compared to absolute terms, as well as data coding inconsistencies between and within jurisdictions, pose challenges in results comparisons. Methodological uncertainties hinder our ability to compare estimates of pandemic-related impacts on outcomes, using pre- and post-pandemic data, due to the diversity of statistical methods employed and variations in the timing and nature of the social distancing measures that were implemented. Importantly, even though we endeavored to harmonize findings by presenting studies that used similar indicators in infographics, there were high levels of heterogeneity in the approaches used to measure substance use outcomes across studies which made comparisons difficult. These graphical representations allowed us to identify trends that should be further examined and provide valuable information to support future meta-analyses using compatible measurements. Most promisingly, quantifiable relative changes in usage between drug types, changes in access measures for harm reduction services , and analyses of non-fatal overdoses as reflective of ED/ER usage changes due to pandemic behavioural responses would be candidate meta-analyses outcomes for future work.As seen with most other non-cohort studies, ascertaining timely and reliable data on various dimensions of substance use can be exceptionally difficult, thus explaining the predominant use of medical records or cross-sectional survey data as shown by our review. Although such data can provide a good basis for decision-making during urgent times, an over-reliance on healthcare system data may hinder the collection of other important substance use-related outcomes. In addition, our ability to make inferences about the precise impacts of the COVID-19 pandemic on aspects relating to substance use are often limited by the reliance on a pre/post change  study design. As a non-randomized approach, this study design carries the risk of bias as a result of confounding factors that distort the comparability of data collected during the different time periods. For this reason, uncontrolled pre/post change studies are heavily discouraged . HoweverWe characterized different data sources, study designs, and measures used to investigate the impact of COVID-19 on illicit substance use in the US and identified key outcomes that could be further investigated through systematic reviews/meta-analyses. Future research should focus on utilizing longitudinal study designs from pre-existing cohorts and on developing routine health data collection systems  to draw more robust inferences on the magnitude and duration of trends in substance use and enable a more effective public health response during crises.Since the onset of the COVID-19 pandemic, there have been transformative changes in substance use health outcome trends in the United States. Despite these changes, this review finds limited evidence to demonstrate any corresponding changes in frequency, behaviors, and contexts of illicit substance use. The findings reveal a need for improved data collection practices to facilitate the conduct of timely research and formulation of policy responses to mitigate and prevent the health harms associated with evolving substance use contexts.


Immunotherapy drugs are transforming the clinical care landscape of major human diseases from cancer, to inflammatory diseases, cardiovascular diseases, neurodegenerative diseases and even aging. In polygenic immune-mediated inflammatory diseases (IMIDs), the clinical benefits of immunotherapy have nevertheless remained limited to a subset of patients. Yet the identification of new actionable molecular candidates has remained challenging, and the use of standard of care imaging and/or histological diagnostic assays has failed to stratify potential responders from non-responders to biotherapies already available. We argue that these limitations partly stem from a poor understanding of disease pathophysiology and insufficient characterization of the roles assumed by candidate targets during disease initiation, progression and treatment. By transforming the resolution and scale of tissue cell mapping, high-resolution profiling strategies offer unprecedented opportunities to the understanding of immunopathogenic events in human IMID lesions. Here we discuss the potential for single-cell technologies to reveal relevant pathogenic cellular programs in IMIDs and to enhance patient stratification to guide biotherapy eligibility and clinical trial design. Pioneered by anti–tumor necrosis factor (TNF) antibodies (Abs), the introduction of targeted therapies against inflammatory cytokines transformed the clinical outcome of patients with major chronic immune-mediated inflammatory diseases (IMIDs), such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD) –3. More In monogenic forms of IMIDs where disease pathophysiology is predominantly driven by a unique molecular pathway, substantial clinical success has been achieved. This is for instance the case in autoinflammatory diseases driven by sustained activation of the inflammasome, such as familial Mediterranean fever (FMF) or cryopyrin-associated periodic syndrome (CAPS) , 6. SubsIn contrast, the clinical benefits of immunotherapy in polygenic IMIDs have remained limited to a subset of patients. About 30-40% of IBD patients do not respond or end up not responding to anti-TNF Abs , 11. OnlThe sequencing revolution that has occurred over the last decades has propelled enormous enthusiasm and surge to identify the underlying genetic drivers of diseases. Genetic profiling has led to significant advances in the identification, understanding and treatment of monogenic immune diseases such as primary immunodeficiencies (severe combined immunodeficiency (SCID), common variable immunodeficiency (CVID) etc…), auto-inflammatory diseases , CAPS, Blau syndrome etc…) and monogenic autoimmune diseases , immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX), (cytotoxic T-lymphocyte-associated protein-4) CTLA4 deficiency etc…) , 14–16. While the clinical benefits of genetic testing are limited to individuals at risk, genome-wide association studies (GWAS) have been extensively used to identify potential mechanisms of diseases in larger patient populations. In most IMIDs, however, only a minor fraction of disease trait variability has been linked to specific genomic loci, and risk gene variants confer small and unpredictable risk of disease occurrence. The role for extragenetic factors has been supported by epidemiologic observations showing discordance of disease expression between homozygous twins, onset frequently occurring during adulthood , and theGenetic studies should by no means be reduced but it is urgent that we enhance our efforts to understand the cellular contribution to disease pathophysiology. Functional genetics can map risk loci or genes to a cellular phenotype but cellular- and tissue-specific effects of gene mutations often lead to distinct disease pathophysiology. This has been well demonstrated in cancer lesions, where loss-of-function or gain-of-function alterations in specific genes can promote tumorigenesis in some tissues while ineffectual in others . For exaImmunologists have excelled in cellular taxonomy  but the nature of the cellular and molecular pathways that lead to human immune diseases remain sparse. Improving the prediction on gene effects and their therapeutic potential will necessarily depend on the understanding of the cellular phenotypes that populate the diseased organs. In that regard, recent data have emerged to support the potential of population-scale single-cell RNA sequencing (scRNA-seq) analyses to explore multiple expression quantitative trait locus (eQTL) in a cell type-specific manner , 27. ComBefore that, however, efforts to improve the access to biological samples and the even rarer access to longitudinal sampling of human lesions during disease progression or during treatment must be initiated, as discussed below.Most drug targets fail to succeed when evaluated in phase 3 trials, hence accounting for both an important economic burden and a distraction for physicians and scientists, which adversely impacts clinical care. The reasons leading to failure are numerous and include factors such as safety issues, insufficient enrollment, suboptimal study design and chiefly, the inability to demonstrate drug efficacy . In factHigh dimensional mapping strategies now provide with unique opportunities to thoroughly describe the sequence of immunopathogenic events developing along disease course and therapeutic intervention in human lesions . AmbitioLongitudinal profiling studies, in particular, require that ethically-acceptable conditions be defined and properly framed by ethical review boards. While repeated sampling in organs that would put the patient at risk with no direct clinical needs is simply not ethically acceptable, it remains possible in tissues such as the skin or the digestive tract. In addition, because histology-based decisions are gaining prominence in the therapeutic management of patients with IBD or chronic inflammatory lesions of the stomach for instance , 35, thiEmpowered by the progress of next generation sequencing (NGS) technologies, genomic assays are currently being used to search for molecular patterns that can stratify patients’ lesions beyond standard histological assays. Genomic assays that include transcriptomic information contributed to the identification of several molecular signatures of IMIDs, some of which have been successfully correlated to distinct clinical outcomes –48. Strahi monocytes, plasmacytoid dendritic cells (pDCs), plasma cells (PCs), and cytotoxic natural killer (NK) cells in patients with the highest disease activity  cells organizing B-cell responses and antibody production within pathologically inflamed synovial tissues  provides an unbiased method to identify cellular subtypes and/or states defined by distinct molecular programs based on the analysis of thousands of genes, which has the potential to reveal scarce cellular processes relevant to IMIDs pathogenicity. Using scRNA-seq, the profiling of peripheral blood mononuclear cells (PBMCs) from SLE patients allowed to identify the expansion of populations of IFN-stimulated genes (ISG)activity . An elevactivity , 43. In g layers , 55. Buiroblasts . Using c tissues . Throughectively , 59. In ectively . In turnectively .Bacteroidales shifted their cytokine responses from the production of immunoregulatory IL-10 toward IL-17 during disease flares (+-scleroderma-associated fibroblasts (ScAFs) from healthy to pathological states in the skin of patients with autoimmune systemic sclerosis (SSc)  sequence analyses allow to generate valuable information about T cell clonal fates, dynamics and effector functions. Such approaches revealed the expansion of T cell clones with cytokine signatures relevant to type 2- and type 3-mediated pathogenicity in lesions of AD and psoriasis respectively . In Crohe flares . The comis (SSc) . InteresDisease-associated cell subtypes do not act in isolation but involve cellular interactions between distinct immune and non-immune cell populations, which together participate in shaping the organization of pathophysiological responses in lesions. Profiling cellular interactions is only possible using technologies that provide single cell resolution at scale. We and others recently identified cellular interactions that correlated with disease course , 63–69. The unique potential for single-cell profiling strategies to identify distinct cellular and molecular organization among clinically and histologically homogenous patients provides enormous opportunities to transform clinical care Figure 1A major goal of single-cell profiling and multiplex imaging technologies is to associate clinically relevant outcomes with cellular signatures. scRNA-seq enables to agnostically resolve the organization of complex cellular signatures in tissue lesions. Cost and labor constraints, however, restrain its application to small cohorts of patients. Because the distributions of clinical and/or demographic parameters can be highly intercorrelated, it is necessary to disentangle confounding factors in order to extract clinically meaningful molecular signatures, which is only possible with large cohorts. We suggest to overcome these limitations using a 2-steps strategy. In a “molecular discovery” phase, relevant cellular signatures are first identified by applying high-resolution single-cell profiling to a small cohort of patients thoroughly selected by experienced clinicians  for his critical reading of the manuscript and suggestions. JM is supported by NExT “Junior Talent”, ANR JCJC (ANR-20-CE17-0009) and ARC Programmes labellisés (PGA). NC is supported by a Fondation pour la recherche médicale (FRM) PhD-fellowship (FDM202106013475).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Neoadjuvant chemotherapy (NACT) indications have expanded from inoperable locally advanced to early-stage breast cancer. The pivotal role of magnetic resonance imaging (MRI) with morphological and functional modalities is making headway in the assessment of tumor size in the staging, residual tumor and prediction of response. Radiomics and radiogenomics MRI applications in the setting of the prediction of the response to NACT in breast cancer are continuously increasing. Nevertheless, there are still controversies regarding the indication of MRI in this setting. Given numerous publications and clinical trials regarding this field, we sought to summarize this complex literature to help clarify the role of MRI in evaluating the tumor size in the staging, response assessment, and surgical planning in early-stage breast cancer patients receiving NACT.Breast cancer (BC) is the most common cancer among women worldwide. Neoadjuvant chemotherapy (NACT) indications have expanded from inoperable locally advanced to early-stage breast cancer. Achieving a pathological complete response (pCR) has been proven to be an excellent prognostic marker leading to better disease-free survival (DFS) and overall survival (OS). Although diagnostic accuracy of MRI has been shown repeatedly to be superior to conventional methods in assessing the extent of breast disease there are still controversies regarding the indication of MRI in this setting. We intended to review the complex literature concerning the tumor size in staging, response and surgical planning in patients with early breast cancer receiving NACT, in order to clarify the role of MRI. Morphological and functional MRI techniques are making headway in the assessment of the tumor size in the staging, residual tumor assessment and prediction of response. Radiomics and radiogenomics MRI applications in the setting of the prediction of response to NACT in breast cancer are continuously increasing. Tailored therapy strategies allow considerations of treatment de-escalation in excellent responders and avoiding or at least postponing breast surgery in selected patients. Breast cancer (BC) is the most common cancer among women worldwide and represents one of the leading causes of mortality and morbidity. In the last decade, the survival rate of BC has been rising thanks to screening and improvements in pharmacological and surgical treatments .Neoadjuvant chemotherapy (NACT) is a systemic treatment administered before surgery . The devNACT is currently also used for patients with early disease, categorized as operable, showing clinically node-negative breast cancer with an unfavorable profile, for whom an adjuvant systemic therapy is recommended . NACT haThe definition of pathological complete response (pCR) varies; for the purpose of designing trials, the FDA recognizes either the absence of invasive cancer in the breast and in the axillary lymph nodes (ypT0/is N0) or invasive cancer and in situ cancer (ypT0 N0) after NACT .Achieving pCR has been proven to be an excellent prognostic marker indicating better disease-free survival (DFS) and overall survival (OS) compared with patients still showing residual invasive disease . The I-SBreast imaging in the pre-treatment evaluation is mandatory to assess the extent of disease and guide biopsies for confirming pathology. It should include a digital mammogram , breast and axillary hand-held ultrasound (HHUS) and in selected patients, magnetic resonance imaging (MRI). Histopathologic confirmation by core biopsy under imaging-guidance and assessment of estrogen receptor (ER), progesterone receptor (PR), HER2, and ideally Ki-67 must be obtained before initiating treatment .MRI is the most accurate method for assessing tumor size in the staging and response assessment after NACT ,14. AccoGiven numerous publications and clinical trials in this field, we sought to summarize this complex literature to help clarify the role of MRI in evaluating the tumor size in the staging, response, and surgical planning in early-stage breast cancer receiving NACT.Relevant papers were found through computerized English-language literature searches of MEDLINE and PubMed Databases in the last 10 years up to May 2022, including the terms: “tumor size”; “breast cancer”; “magnetic resonance imaging”; “neoadjuvant chemotherapy”. In addition, articles were traced through references listed in relevant papers and in previous reviews.The goals of pre-therapy imaging are to determine local-regional staging and to check the contralateral breast. The extent of residual disease, displayed by imaging after NACT, aids in guiding surgical planning.Data from single-institution and ,18 multiA meta-analysis of 44 studies between 1990 and 2008, including 2050 patients, found that MRI after NACT generally had high sensitivity (83–87%) and heterogeneous specificity (54–83%) in detecting residual disease . This mer = 0.77). In contrast, masses with ill-defined margins had a lower correlation (r = −0.19). Although a decrease in tumor size indicates treatment response, there is no correlation between the change in the extent of the calcifications after NACT and pathologic complete response [Data about mammography and HHUS in the setting of response to NACT are variable. The initial mammographic appearance of the tumor affects the accuracy of mammography in the evaluation of residual tumor size. Huber et al.  found thresponse .Reportedly, HHUS is more accurate than mammography in estimating residual tumors. No difference in accuracy was found between MRI and HHUS, even though HHUS is deemed unsuitable for monitoring tumor size because it is subjective and operator dependent . Few stuContrast-enhanced mammography (CEM) allows the evaluation of vascularized lesions such as with breast MRI . CompareIt is now generally acknowledged that there are various subtypes of breast cancer, each with a unique pathophysiology, prognosis, and course of treatment. Four primary molecular subtypes of invasive breast cancer have been found by gene expression profiling and are identified in clinical care based on immunohistochemistry: luminal A, luminal B, HER2 positive, and triple negative (TN) . Many stLuminal A is the most prevalent type of breast cancer, commonly managed by surgery and hormone therapy. Even though they are ER-positive, luminal B tumors are more resistant to endocrine therapy, having in common many molecular characteristics with ER-negative subtypes . LuminalHER2 positive cancers have several MRI characteristics that are comparable to those of luminal tumors; heterogeneous non-mass enhancement, perilesional oedema, and higher apparent diffusion coefficient (ADC) values than other subtypes, are a few characteristics that may indicate HER2 positive cancers .On MRI, TN breast cancer is the most distinct tumor. Masses with smooth margins, round or oval shape, high T2 signal intensity, rim enhancement, and intratumoral necrosis are suggestive of histopathological TN breast cancer ,48.Similar MRI appearance can be seen in uncommon histological forms of invasive breast cancers, representing a diagnostic challenge, e.g., mucinous carcinoma that exhibits high T2 signal intensity due to its predominant mucin component, but it often shows a persistent type of curve .Enhancement kinetics showed poor predictability of molecular subtypes. Poor prognosis predictors are early maximum enhancement, wash-out in the delayed phase and rim enhancement and they are related with higher histologic grade, positive Ki-67, and negative ER status .A correlation was found between the type II enhancement curve and luminal A tumors, possibly explained by limited neo-angiogenesis compared to other molecular subtypes . Table 1MRI accuracy has different discrepancy according to histological subtype; data indicate that infiltrating ductal carcinoma has a significantly lower mean discrepancy when compared with infiltrating lobular carcinoma .Early prediction of pCR following NACT could be an important tool for personalized medicine, allowing the selection of patients eligible for NACT .Many studies have demonstrated how the evaluation of multiparametric breast MRI obtained before the start of NACT can predict which cancer will achieve pCR, through the analysis of morphological and functional features and the application of advanced imaging and artificial intelligence (AI) techniques.Thus, the challenge remains the early and accurate predictions of NACT response.Among the morphologic MRI features, an expansively growing carcinoma forming a relatively well-defined round/oval or lobulated mass is often associated with low rates of pCR, suggesting that careful attention should be paid during the course of the therapy in these types of tumors . In patiIn a study by Thompson et al. , the autSome authors have suggested a correlation between the background parenchymal enhancement (BPE) and tumor response after NACT. BPE is defined as normal enhancement of the fibro-glandular tissue on contrast-enhanced (CE) MRI and is thought to be associated with poorer prognosis: several studies found a significant association between high BPE on baseline MRI and worse recurrence free survival (RFS) ,59. ThisIn a study by Preibsch H et al. , the autFunctional imaging sequences, such as DWI-MRI and CE-MRI, allow the analysis of biological characteristics of tumors, such as cellularity and neo-angiogenesis, by assessing cellular density of a tissue, quantified using ADC map. However, the analysis of the ADC value on pretreatment and pCR imaging remains controversial, since some tumors may have low cellularity  due to local necrosis secondary to hypoxia or local fibrosis or desmoplastic components . Accordi2/s based on the ROC curve analysis  [−3 mm2/s using a high b-value of 750 s/mm2, with a sensitivity of 94% and a specificity of 71% [In one study, the best pre-treatment ADC cut-off value to distinguish between responders and non-responders was found to be 0.55 mmy of 71% .Conversely, some other authors found no significant difference in the mean ADC value between responders and non-responders in the general population ,68. HoweSome works investigated total choline (TCho) levels in breast cancer through Magnetic Resonance Spectroscopy (MRS). TCho is a biomarker of elevated cellular turnover and is thought to be an early imaging predictor of response to treatment but its prognostic value remains unconfirmed ,71. HoweDifferent cancer subtypes show different pCR rates with higher rates in HER2 positive and TN breast cancer , reachinHowever, TN breast cancer is more aggressive than other subtypes and associated with a higher rate of relapse and a lower rate of OS ,71.TN breast cancer is more likely to display intramammary edema, resulting from lymphovascular invasion. Peritumoral edema occurs due to local reaction of the surrounding tissue to the tumor, pre-pectoral edema is determined by marked lymphovascular invasion, whereas subcutaneous edema reflects the lymphovascular invasion. The correlation between response to NACT and the presence of edema is controversial. Bae et al.  revealedAnother common finding in TN breast cancer is intratumoral necrosis, probably due to the increased mitotic activity, which is often associated with poor or no response to NACT . HoweverIn addition, it has been shown that TN breast cancer presenting as an irregular mass is less likely to respond to NACT than that with other appearances .In a study by Li et al. , a nomogRadiomic analysis is a method to extract quantitative information from each voxel of radiological images, to predict clinical data. Radiomics applications are constantly increasing given their ability in the prediction of pCR in various cancer types, including breast cancer . In a reAs stated before, the definition of pCR is still the object of debate with some trials defining it as the absence of both in situ and invasive cancer while others considering only the invasive component . The accpCR has been proven to be a good prognostic marker to predict long-term survival in breast cancer ,89; therIn order to reach the maximum surgical advantage from NACT, it is crucial to correctly evaluate the tumor response and residual disease prior to surgery. The Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 guidelines  recommenDifferent shrinkage patterns are seen in breast cancer after NACT, including no residual tumor (pCR) , concentKim et al.  found thSome authors tried to quantify the degree of residual enhancement by using the lesion to background parenchymal signal enhancement ratio (SER), which has been found to increase the specificity in detecting residual tumor rather than using size criteria alone (with SER ≤ 1.6 indicating pCR) .It has been suggested that the type of chemotherapy agent should be considered when using MRI in the evaluation of tumor response. Patients treated with taxane-containing regimens  and in HMRI also allows in vivo evaluation of treatment efficacy, which may permit changing the treatment approach if the tumor is not responding. Some studies reported the ability of MRI to assess the efficacy of treatment early, after 1–2 cycles of NACT, based on imaging data of volumetric changes, kinetic analysis, both  and metaOverall, based on its ability to show the extent of tumor after NACT and increasing numbers of studies demonstrating early response prediction, MRI is a useful tool in helping the multidisciplinary team to choose the most suitable therapeutic planning, especially surgical treatment aimed at local disease control. However, up to now, no randomized control trial has demonstrated that using MRI for monitoring the response to NACT increases the rate of breast conserving surgery. On the other hand, also in the neoadjuvant setting, MRI is considered useful for appropriate surgical decision making likewise to preoperative MRI in the absence of NACT .Breast conserving surgery (BCS) is nowadays the standard surgical treatment for most early-stage breast cancers (EBC) and locally advanced breast cancers after good NACT response ,104,105.Although the diagnostic accuracy of MRI has been shown repeatedly to be better to conventional examinations in assessing the extent of breast disease, literature does not agree that the routine use of MRI, as part of pre-operative staging even after NACT, leads to an improvement in surgical outcomes. For this reason, most guidelines and recommendations suggest that preoperative MRI should only be used in selected cases ,113,114.The fear that MRI may lead to an increase in the rate of mastectomies, beyond what is necessary, has not yet been completely dispelled .However, in recent years, studies with large numbers of patients have shown that a small increase in mastectomy rates can be well compensated by a reduction in recurrences; for example, Sardanelli et al.  performeNot many studies have evaluated the long-term outcome of presurgical MRI, and most studies do not indicate a decrease in metachronous second breast tumors . There iIn patients with excellent response to NACT and subsequent pCR, it is oncologically safe to de-escalate treatment ,116,117.The data show that patients with breast cancer and pCR to NACT are very unlikely to face distant recurrence; in contrast, minimal residual pathology with subsequent risk of local relapse is still possible. Studies are currently underway to identify pCR, using barely invasive biopsies in women who have had partial/complete radiologic response after treatment, in order to stratify low-risk patients .In addition, there may also be subset of patients who do not benefit from surgery after treatment; however, there is currently insufficient scientific evidence. Therefore, several questions remain unsolved, and it remains a great challenge for the future to conduct high-quality clinical trials that can answer these questions .We are aware of the fact that this narrative review might have some limitations: it is non-systematic and only English language papers have been considered. Moreover, the rapid information produced in the literature on this topic might have caused some missing new relevant paper. However, the overall evaluation points out that indications for neoadjuvant systemic treatment have growth from inoperable locally advanced to early-stage breast cancer. MRI plays a pivotal role and with morphological and functional modalities is making headway in tumor size evaluation, in the staging and in response prediction. Radiomics and radiogenomics MRI applications in the setting of breast cancer NACT response prediction are continuously increasing. Tailored systemic therapy strategies allow considerations of treatment de-escalation in excellent responders and avoiding or at least postponing breast surgery in selected patients.


Decreasing biotic diversity with increasing latitude is an almost universal macroecological pattern documented for a broad range of taxa, however, there have been few studies focused on changes in gut microbiota (GM) across climatic zones.Using 16S rRNA amplicon profiling, we analyzed GM variation between temperate (Czechia) and tropical (Cameroon) populations of 99 passerine bird species and assessed GM similarity of temperate species migrating to tropical regions with that of residents/short-distance migrants and tropical residents. Our study also considered the possible influence of diet on GM.We observed no consistent GM diversity differences between tropical and temperate species. In the tropics, GM composition varied substantially between dry and rainy seasons and only a few taxa exhibited consistent differential abundance between tropical and temperate zones, irrespective of migration behavior and seasonal GM changes. During the breeding season, trans-Saharan migrant GM diverged little from species not overwintering in the tropics and did not show higher similarity to tropical passerines than temperate residents/short-distance migrants. Interestingly, GM of two temperate-breeding trans-Saharan migrants sampled in the tropical zone matched that of tropical residents and converged with other temperate species during the breeding season. Diet had a slight effect on GM composition of tropical species, but no effect on GM of temperate hosts.Consequently, our results demonstrate extensive passerine GM plasticity, the dominant role of environmental factors in its composition and limited effect of diet. Microbial communities associated with animal hosts have a marked effect on the host’s physiology and immune system . In the via selection of host phenotypic traits that affect colonization and proliferation of bacteria within the gut. In birds, high temporal stability and predictability of resources in tropical environments are associated with a comparatively higher life expectancy and a slower pace of life compared to phylogenetically related species inhabiting temperate climatic zones  and willow warbler (Phylloscopus trochilus).In this study, we analyzed faecal microbiota, which has been shown to be a good proxy for avian GM , with san = 148 samples and 19 species; n = 257 samples and 33 species; Temperate species were categorized as trans-Saharan migrants with wintering grounds in tropical sub-Saharan Africa . Primers covering the V3-V4 variable region on bacterial 16S rRNA  were used during the PCR step . For the2 Using the Decontam package  of individual OTUs in each sample as response variables. The models estimated correlation due to host phylogeny, while systematic variation among species was modeled via random effects. All models, except those focused on OTU abundance, were assumed to have a Gaussian distribution. Models based on OTU abundance were assumed to have a negative binomial distribution, while the log-scaled total number of sequences per sample was considered as an offset to account for uneven sequencing depth. To achieve convergence between the OTU-specific models, each model was only fitted for OTUs detected in >20 samples and represented by >500 sequences in total. Significance testing associated with GLMMs was based on type II sum of squares, meaning that the effect of climatic zone, migration and season was adjusted for the effect of diet and vice versa. In the case of OTU-level analyses, false discovery rates . All statistical analyses were undertaken using the R statistical environment v.4.0.3 . Specifically, for each pair of host categories , we extracted two vectors of GM dissimilarity corresponding either to different host species from the same category or to different host species from different categories. Dissimilarities for same host species pairs were not considered. Next, we calculated the mean difference between these two vectors and corresponding 95% bootstrap-based confidence intervals using the two.boot function in the R package simpleboot , with no significant difference in their values . The same held true for the comparison of rainy season tropical samples with the two groups of temperate hosts . This suggests, that tropical environment does not directly affect GM of trans-Saharan migrants during their breeding season.Differential abundance analysis for the 121 dominant OTUs  identified 40 OTUs  whose abundances were affected by climatic zone, migration or a variation between dry vs. rainy seasons. More specifically, 19 OTUs in tropical hosts varied between the dry and rainy seasons and the abundance of three OTUs varied between trans-Saharan migrants and temperate zone residents/short-distance migrants. In addition, there was pronounced variation between temperate and tropical hosts sampled both during the dry  and rainy seasons  and the willow warblers (P. trochilus) in the temperate zone during their breeding season  and in the tropical region during their migration and wintering period . PCoA suggested a dramatic effect of tropical and temperate environments on GM composition in both species. PCoA ordination showed that, while GM samples collected at their breeding grounds showed perfect overlap with other temperate zone passerines, their GM changed dramatically in the tropics. The direction of this change was congruent with the overall direction of the difference between temperate species and tropical passerines from the dry season  to assess the actual effect of climatic zone on GM. Second, we conducted a comparative analysis using GM sampled during the breeding season for temperate passerines that varied in migration behavior, allowing us to evaluate long-lasting pervasive effects of migration behavior on GM.Staphylococcus OTU, which contributed most to GM compositional change between wintering and breeding grounds, was also more abundant in tropical species during the dry season than during the rainy season. Altogether, our data suggest that environment is the main driver of GM variation between wintering and breeding grounds in trans-Saharan migrant species. Consistent with our results, several previous studies have reported variation in GM composition between breeding and wintering grounds, or during spring and autumn migrations in other migratory birds (The GM of the two species of trans-Saharan migrants (collected at breeding grounds in the temperate zone and at dry season in the tropics during their wintering period) exhibited substantial differences in composition, but not in alpha diversity. According to PCoA sample ordination, these changes corresponded with overall GM differentiation between temperate zone passerines and tropical passerines sampled during the dry season, but not the rainy season. Moreover, a ry birds . HoweverIn addition to spatially diversified pools of environmental microbes interacting with the GM of long-distance migrants, behavioral, physiological and ecological adaptations associated with migration  could alLactococcus) of three OTUs over-represented in the subset of all trans-Saharan migrants at their breeding grounds (compared to other temperate zone species) exhibited higher abundance in tropical hosts during the dry season than in temperate residents/short-distance migrants. Finally, two OTUs, exhibiting a striking increase in abundance in garden and willow warblers at their wintering grounds, did not vary between trans-Saharan migrants and other temperate passerines at their breeding grounds. Altogether, the results indicate that GM acquired at wintering grounds and during migration rapidly converge to a GM typical for the temperate zone following their spring arrival at the breeding grounds. This is in line with our previous study demonstrating a low level of GM stability over time in another trans-Saharan migrant, the barn swallow , tended to decline gradually after returning from their wintering grounds.According to our comparative analysis comprising 19 species of trans-Saharan migrants and 33 temperate residents/short-distance migrants, however, the GM of trans-Saharan migrants during the breeding season showed only a small, though still significant, difference to that of other temperate species. Furthermore, our data suggest that these differences are unlikely to be caused by bacteria incorporated into GM during overwintering and migration as alpha diversity of trans-Saharan migrants was not increased compared to other temperate zone passerines and GM composition did not show a lower dissimilarity than tropical hosts. Moreover, only one  that prefer energy-rich substrates and are capable of fermenting carbohydrates under anoxic conditions , and Charles University in Prague (4789/2008-0).JKr and TA: study design. TA, MT, OT, TK, and OS: field sampling. LS and JKu: laboratory analysis. JKr and LS: data analysis and manuscript drafting. TA, JKr, and JKu: funding. All authors provided helpful comments and recommendations and approved the final version of the manuscript.


In pulmonary diseases, new therapeutic strategies have emerged that are aimed at restoring functional lung tissue rather than alleviating symptoms. These strategies focus on tissue regeneration using stem cells and/or their derivatives or replacement of dysfunctional tissue using biomedical engineering. Animal health can directly benefit from regenerative therapy strategies and also serve as a translational experimental model for human disease. Several clinical trials have been conducted to evaluate the effects of cellular treatment on inflammatory lung disease in animals. Data reported to date show several beneficial effects in  Several chronic respiratory diseases in humans and animals remain incurable. Treatments have been relatively successful in relieving some symptoms, but they all ultimately lead to a poorer quality of life and are one of the leading causes of death worldwide . IntenseThe lung has an exceptional ability to respond and regenerate after the tissue injury , 9, 10. Although inflammation is the reason for damage to the airways, it is also critical for initiating tissue regeneration and restoration. Inflammatory cells flooding the airways are important for phagocytosis and for stimulating resident progenitor cells through secreted cytokines and growth factors. Some resident cell populations do not appear to exist in a healthy lung, but emerge only in response to lung injury. A more detailed knowledge of this relationship will likely enable new therapeutic options to stimulate lung regeneration and self-repair .®  search engine (https://pubmed.ncbi.nlm.nih.gov/), evaluating reports from January 1, 1990, to October 31, 2022. Reference lists of relevant articles were also reviewed to find additional studies.An online literature search was performed using the PubMedex vivo (exogenous) cultivation of SC and their application in patients  play a leading role. Stem cells are undifferentiated cells capable of self-renewal and transformation into other cell types . Traditipatients , 48, 49.patients , 48, whiHeterogeneous endogenous stem cells [cells capable of long-term self-renewal and differentiation into other progenitor cells or tissue-specific cells ] and proBasal epithelial cells represent a population of stem/progenitor cells from which Club cells (formerly known as Clara cells) and ciliated cells can develop , 52. TheResident stem cells, which are thought to share several properties with bone marrow-derived mesenchymal stem cells (BM-MSC), have also been found in the lung , 55–57. Attempts have also been made to derive the phenotype of structural lung cells for pulmonary vascular regeneration from adipose or bone marrow tissue or from embryonic SC. Despite the ability of SC to differentiate into lung cell types, results of such studies remain controversial because of inadequately derived or described methods .The therapeutic potential of exogenous SC has been repeatedly noted in relation to their immunomodulatory effects. Their complex immunomodulatory role results from their interaction with local immune cells and paracrine signaling, leading to a reduction in proinflammatory stimulus and thus less tissue damage , 60–63. in vitro-grown tissue may bridge the time to lung transplantation or serve as a definitive therapeutic modality.Pathologic changes in diseased lungs may progress to the point where cell therapy and stimulation of tissue regeneration alone are insufficient and tissue replacement is required to restore lung function. Suitable lung donors are not always available, or lung transplantation is contraindicated ; therefoin vitro and mimicking their function is a major challenge that requires a high degree of cell specialization and complex tissue architecture  anatomic architecture . Generatitecture , 70, 71.itecture .Most preclinical studies have used biologically derived models or synthetic scaffolds seeded with an appropriate cell source to regenerate functional lung tissue , 7. Hybrin vivo and the properties of gene expression can be obscured or lost in more commonly used in vitro 2D cell culture systems , messenger RNA, proteins, and mitochondria. Extracellular vesicles are broadly classified into exosomes , microvesicles , and apoptotic bodies  . The useConfirmation of the functionality of ECV therapy requires that the therapeutic effect occurs without intercellular contact and that this is not achieved by ECV-unrelated soluble paracrine factors . ExtraceNumerous studies have demonstrated the benefits of systemic administration of ECV in mitigating allergic airway hyperreactivity and resulting inflammation and tissue remodeling , 31. ExtIn addition to the cell-free regenerative medicine options described above, several indirect therapeutic options have been described to stimulate local cells and tissue regeneration in the airways. For example, all-trans-retinoic acid, a derivative of vitamin A (retinol), has been described as a possible candidate to promote alveologenesis , 82, 83.Benefits of cell treatments have been reported for the treatment of asthma; experimentally in mouse models , 26–31 aIngenito et al.  investigTreatment with SC also significantly affects inflammatory responses and lung tissue regeneration in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) , 88–91. In addition to cell therapy, treatment with ECV has also successfully treated acute airway inflammation caused by viral infection. Khatri et al.  investigFurther evidence from appropriately designed clinical trials is needed before regenerative therapy is considered an accepted therapeutic modality in respiratory medicine. To date, the use of SC or ECV for the treatment of respiratory disease has consistently been described as relatively safe after local and systemic application. Apart from mild local reactions after administration of cells of allogeneic origin, no severe adverse events have been observed , 93–96. It is also important to focus on a detailed understanding of the functional heterogeneity of each cell type in the respiratory system and the development of protocols for targeted cell differentiation and maturation . This isAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Hydrogels are thought of as unique polymers utilized to build new materials, and two key factors that impact their features are their hydrophilicity and the degree of cross-linking of the polymer chains. An injectable hydrogel is based on the hypothesis that certain biomaterials can be injected into the body as a liquid and progressively solidify there. The scientific research community was intrigued and interested by its discovery. The hydrophilic polymers that are used to make hydrogels can typically be split into two groups: natural polymers derived from tissues or other sources of natural materials, and synthetic polymers produced by combining principles from organic chemistry and molecular engineering. A variety of organic and synthetic biomaterials, such as chitosan, collagen or gelatin, alginate, hyaluronic acid, heparin, chondroitin sulfate, polyethylene glycol, and polyvinyl alcohol, are used to generate injectable hydrogels. A promising biomaterial for the therapeutic injection of cells and bioactive chemicals for tissue regeneration in both dentistry and medicine, injectable hydrogels have recently attracted attention. Since injectable scaffolds can be implanted with less invasive surgery, their application is seen as a viable strategy in the regeneration of craniofacial tissue. Treatment for periodontitis that effectively promotes periodontal regeneration involves injecting a hydrogel that contains medications with simultaneous anti-inflammatory and tissue-regenerating capabilities. The advantages of injectable hydrogel for tissue engineering are enhanced by the capability of three-dimensional encapsulation. A material's injectability can be attributed to a variety of mechanisms. The hydrogels work well to reduce inflammation and promote periodontal tissue regeneration. Whichterler and Lim made the first discovery of hydrogels in 1960 [D networkAn injectable hydrogel is based on the concept that some biomaterials can be injected into the body as a liquid and then solidify in place. Injectable hydrogels have generated a lot of attention in the areas of medication delivery, tissue engineering, and dermal fillers because they have the necessary physicochemical qualities to be injected in situ into the body . One espHistory of hydrogelsThe first hydrogel poly-2-hydroxyethyl methacrylate (PHEMA) was created and described in 1960 by Whichterler and Lim. They used it to make moisture-absorbing contact lenses. Modern hydrogels are demonstrated and resembled by their 3D crosslinking structure. The scientific research community was intrigued and interested by its discovery. Then, in the 1980s, Lim and Sun created calcium-alginate gel composites for islet-droplet microcapsule cell embedding [According to Buwalda et al., there have been three distinct generations of hydrogels. The first generation of hydrogels mostly consisted of gels with diverse crosslinking techniques created by chemically altering a monomer or polymer using an initiator. After this time, in the 1970s, the significance of hydrogels increased to a new level as stimuli-responsive properties were incorporated into the hydrogels, allowing second-generation hydrogels to react to a variety of highly specific stimuli, including changes in pH, temperature, or the concentration of certain biomolecules in a solution. The focus switched to the creation of stereo-complexed biomaterials and hydrogels joined through physical interactions in the third-generation hydrogels. These changes prompted scientists to focus their efforts more intently on creating the current "smart hydrogels," which can be tailored to acquire specific qualities like stimulus responsiveness and adjustable mechanical and other physicochemical properties [Building blocks for the preparation of injectable hydrogelsA variety of materials are used to create injectable hydrogels. Generally speaking, there are two types of hydrophilic polymers that are utilized to make hydrogels: natural polymers taken from tissues or other natural sources, and synthetic polymers created utilizing organic chemistry and molecular engineering concepts. Building blocks of synthetic and biocompatible natural polymers are used to create the injectable hydrogels shown in Table Types of injectable hydrogelsNatural and synthetic biomaterials, such as chitosan, collagen or gelatin, alginate, hyaluronic acid, heparin, chondroitin sulfate, polyethylene glycol (PEG), and polyvinyl alcohol (PVA), are used to form injectable hydrogels. Chemical techniques that use covalent crosslinking produced by enzymes, physical techniques that use weak secondary forces, chemical techniques that use photo-cross-linking, physical techniques that use Michael addition, and chemical techniques that use click chemistry can all be used to create injectable hydrogels that are ion-sensitive, pH-sensitive, or temperature-sensitive .Chitosan-Based HydrogelsChitosan is a cationic polymer consisting of glucosamine and N-acetylglucosamine that is found naturally. Because of its biocompatibility and biodegradability, chitosan is frequently used in the pharmaceutical and medical fields. Because of the many amino groups on its backbone, chitosan is a great choice for creating injectable self-healing hydrogels based on imine linkages .Phosphate-Based HydrogelsA fresh, injectable oligo-polyethylene-glycol fumarate (OPF) gel for bone healing with continuous release of phosphate ions, improved electrical conductivity, and mechanical strength. The mechanical stability and electrical conductivity of gel formulations were shown to be improved by a carbon nanotube nanocomposite, and the inclusion of two-dimensional (2D) black phosphorus nanosheets allowed a continuous release of phosphate ions by environmental oxidation .Alginate-Based HydrogelsAlginate hydrogels with biomedical uses fall into two categories: "Physical" or "reversible" gels are held together by molecular entanglements, hydrophobic forces, and ionic or hydrogen bonding, as opposed to "chemical" or "permanent" gels, which are produced when stable covalent connections crosslink networks. Alginate hydrogels are excellent alternatives for drug carriers because of their high water content, nontoxicity, soft consistency, biocompatibility, and biodegradability. They can transport low-molecular-weight drugs as well as macromolecules like proteins and DNA either sustainably or locally .Hyaluronic Acid (HA)-Based HydrogelsBecause it may imitate tissue ECM and has the ability to influence cell behavior during tissue regeneration, such as cartilage and pulp regeneration, HA has been widely used to make injectable hydrogels. Since HA's molecular structure contains a variety of primary and secondary hydroxyl and carboxyl groups, a bifunctional small-molecule crosslinking agent can also be utilized for this purpose. This results in a HA hydrogel with the optimum composition, shape, hardness, and biological activity .Collagen (col)- or Gelatin (gel)-Based HydrogelsDue to their high levels of biocompatibility, biodegradability, bioactivity, and diversity, gelatin methacryloyl (GelMA) hydrogels are frequently employed for tissue healing. By adding double bonds to the gelatin polymer chains, which under photoinitiation quickly form hydrogels, GelMA hydrogel is created. Lithium acylphosphinate salt (LAP), a blue light initiator, facilitates preparation and speeds up the gelation process. It poses no threat. Injectable hydrogel made from GelMA hydrogel works well and may be molded using 3D printing .Fibrin-Based HydrogelsKnown as a helpful cell-transplantation matrix, they can improve cell adhesion, proliferation, differentiation, and migration in a 3D scaffold. Fibrin is a naturally occurring fibrous protein implicated in blood clotting. Scaffolds have been constructed using fibrin, either by itself or in conjunction with other substances, for applications involving cartilage tissue engineering. A unique injectable hydrogel system has been created using PEG, stem cells generated from human amniotic fluid, and fibrin-based hydrogels that can promote in situ neovascularization and cause a fibrin-driven angiogenic host response .Elastin-Based HydrogelsFor numerous biological applications, injectable hydrogels produced from ECM proteins like elastin hold considerable promise. The primary problems of these hydrogels are their lack of mechanical strength, use of cytotoxic chemicals, and fixed gelling behavior .Chondroitin Sulfate (CS)-Based HydrogelsThe safety and high biocompatibility of CS, a common material for cartilage tissue engineering scaffolds that exhibits quick gelation, outstanding mechanical capabilities, and delayed degradation qualities, have been thoroughly explored and documented .PEG-Based HydrogelsPEG-based hydrogels and their derivatives have received a lot of attention recently due to their capacity to be well tolerated in vivo in the context of drug administration and tissue engineering applications. Because they may be administered easily and painlessly by injecting low-viscosity precursor polymer solutions, injectable, in situ gelling counterparts improve the potential applications of these hydrogels .PVA-Based HydrogelsFor the in situ production of hydrogels under physiological conditions, new PVA compounds with a variety of pendant chemoselective characteristics have been developed. For the first time, PVA was modified by adding thiol, cysteine 1,2-aminothiol, and aminooxy side chains by direct carbamate connections from protected nucleophilic functionalities to the hydroxyl groups of PVA .Shape Memory (SM) and Self-Healing (SH) HydrogelsBoth SM and SH have their roots in reversible interactions. It is quite difficult to synthesize hydrogels using both SH and SM .Interpenetrating Polymer Network (IPN) HydrogelsTo address the conundrum of choosing between a complex structure and an easy biodegradability, a novel method for creating biodegradable hydrogels with an IPN structure that consists of peptide self-assembling networks and a covalently cross-linked network has been proposed .Double Network (DN) HydrogelsDue to their outstanding mechanical and chemical adaptability, DN hydrogels have become leading contenders for tissue engineering. DN hydrogel formulations combined with processing advancements (such as additive manufacturing and injection) have produced a remarkable set of findings that significantly advance the development of systems that can address the complex environment around tissues and allow for individualized fabrication techniques .Programmable HydrogelsProgrammable hydrogels are hydrogels that have the ability to periodically, reversibly, and sequentially alter their properties and functionalities. Using the aforementioned concepts, programmable hydrogels that are induced to undergo functional changes could be produced for a range of intriguing applications .3D Printed HydrogelsIn tissue engineering and regenerative medicine, 3D bioprinting, one of the most recent biotechnologies, is frequently used to produce complex artificial organ and tissue designs that closely resemble genuine organs and tissues. In order to restore functional and site-specific tissues or organs, bioprinting is the additive deposition of cell-loaded hydrogels in a specified structural framework .Injectability mechanisms of injectable hydrogelsA material's injectability can be attributed to a variety of mechanisms. These mechanisms are divided into three major categories: The first mechanism is in situ gelling liquids - solutions or liquids that normally flow but harden into gels when injected into the body. The second mechanism is injectable gels - even though certain gels are created ex vivo, their shear thinning qualities and capacity to restore their hydrogel shape following relaxing procedures make them suitable for injection. The third mechanism is injectable particles - in addition to the two types of injectable systems previously discussed, the third class of injectable particles also has the ability to be injected when immersed in a liquid phase. Depending on the desired outcomes, particle sizes might be nano-, micro-, or macroscale .Applications of injectable hydrogelsTherapeutic Applications in DentistryA promising biomaterial for the therapeutic administration of cells and bioactive compounds for tissue regeneration in dentistry and medicine is injectable hydrogels. They are suitable for minimally invasive surgical operations in a clinical context because they offer adaptable tissue-like qualities, regulated degradation and release behavior, and the ability to conform to the 3D defect upon gelling. A recent development in tissue engineering used a biomaterial scaffold and bioactive substances to encourage endogenous cell migration and tissue repair, further demonstrating the potential of the "homing" strategy for dentin-pulp and craniofacial regeneration . InjectaTherapeutic Applications in Periodontal RegenerationTreatment for periodontitis that effectively promotes periodontal regeneration involves injecting a hydrogel that contains medications with simultaneous anti-inflammatory and tissue-regenerating capabilities. The hydrogels showed outstanding self-healing abilities, an expedient gelation process, and injectability . To reduInjectable Hydrogel Delivery for Tissue EngineeringThe use of injectable hydrogels as a delivery system for topical and localized medication delivery appears promising. The advantages of injectable hydrogel for tissue engineering are enhanced by the capability of 3D encapsulation. An injectable hydrogel must strike a compromise between mechanical properties, carrier capacity, and processability . It has Future directions and current statusDue to their adaptability, injectable hydrogels, a subset of hydrogel, have drawn significant interest in biological applications. According to reports, the injectable hydrogel can be used in a variety of biomedical treatments, such as tissue engineering for cartilage and bone as well as periodontal implants and submucosal fluid cushions. In addition to being simple to implant, this kind of hydrogel can be customized to fit certain purposes .Due to its potential for less invasive local drug administration, more precise implantation, and site-specific drug delivery into difficult-to-reach tissue regions and into interface tissues, where wound healing takes time, injectable hydrogels have attracted a lot of interest in the biomedical industry. It is still extremely desirable to create an injectable hydrogel with self-healing capabilities for continuous, controlled drug delivery. For prolonged protein release, targeted drug delivery, and tissue engineering, hydrogels, microgels, and nanogels have become efficient and useful platforms because of their high biocompatibility and microporous structure with adjustable porosity in periodontal regeneration. Among other things, the hydrogels are successful in reducing inflammation and promoting periodontal regeneration.


Exosomes are extracellular vesicles produced by various cell types and extensively distributed in physiological fluids. Because of their significant role in cancer progression, they have been a focal point for the novel cancer therapy approach. Exosomes are highly efficient at transporting proteins, RNAs, and small drugs into cancer cells for therapeutic purposes. In addition to their prominent role as potential biomarkers for transporting targeted information from their progenitor cells, exosomes have also emerged as a new avenue for developing more effective clinical diagnostics and therapeutic techniques, also known as exosome theranostics. Lipids, proteins, and nucleic acids transported by exosomes were investigated as potential biomarkers for cancer diagnosis, prognosis, and future cancer treatment targets. The unique mechanism of exosomes and their therapeutic as well as diagnostic uses, also known as theranostic applications of exosomes in malignancies, are discussed in this review. Cancer emerged as a severe hazard to humankind, with rising death rates worldwide . The tumExosomes, being the significant portion of TME, have shown to bear specific activities regarding tumor formation, angiogenesis, progression as well as drug resistance . ExosomeAccording to mounting evidence, exosomes can be released by various cells, including lymphocytes and fibroblasts. Exosomes generated from various cells have distinct properties . Cancer This review seeks to give an insight into exosome dynamics and progression in cancer theranostics, including their relationships, exosome uses, and exosome roles derived from different origins. The latest innovations in exosome technology will aid in developing cancer treatment applications shortly.Extracellular Vesicles (EVs) are Nano-scale membrane vesicles that are actively released by cells. They occur through the outward budding of the plasma membrane or microvesicle pathway or inward budding by the inner body membrane or exosomal pathway. The vesicle formed through outward sprouting of the plasma membrane is called Multivesicular bodies (MVBs). Exosomes are vesicles of endocytic origin. The early endosome is formed by the intrusion of the plasma membrane inwardly, and the limiting layer of the subsequent endosome grows further to produce tiny vesicles, resulting in the development of MVBs. The MVB is distinguished by the formation of Intraluminal Vesicles following invagination of the inner body membrane (ILVs). During their development, cytoplasmic inclusions, transmembrane, and peripheral proteins are incorporated into ILVs. ILVs collected in the MVB lumen have two possible outcomes: a) fusion with lysosomes, causing the contents of the vesicles to break down [Multivesicular Endosomes or Bodies (MVBs or MVEs)], or b) fusion with the plasma membrane, releasing ILVs into the extracellular space as exosomes. On the other hand, direct budding from the plasma membrane forms microvesicles, which sequester sections of the cytoplasm . MicroveMore specifically, the role of various proteins varies in different processes and origins of the exosomes. Leading cargo into ILV involves the ESCRT and other related proteins. ESCRRT includes PDCD6IP , TSG101, HRS, CD9, and CD82, etc., Other proteins include PLG2, DGKα, etc., After MVBs fuse with the cell membrane, exosomes are secreted. This process relies on small GTPases such as RAB27A and RAB27B. RAB7, RAB11, RAB31, and RAB35 in some cells or SNARES family proteins like YKT6, VAMP7, etc., Microvesicles (MVs) represent a relatively heterogeneous population of vesicles formed on the outward germination fission of cell membrane lipid microdomains and regulatory proteins such as ADP ribosylation factor 6 (ARF6). MVs participate in immune modulation by assisting in antigen presentation and transmitting MHC molecules and antigens . It is aExosomes are produced in greater quantities (approximately 10 times) by tumor cells than normal cells, resulting in higher exosome concentrations in the blood of cancer patients . Due to Neovascularization is a well-known biochemical phase in the progression of tumors and metastasis. Both stromal and tumor cells control tumor angiogenesis by producing numerous signaling molecules and proteins . ProangiExosomal VEGF regulates angiogenesis through several pathways . MesenchTumor metastasis begins with tumor cells splitting from the primary neoplasm and traveling to a distant organ location, which entails complicated biological processes. Exosomes promote metastasis by interfering with various cellular pathways . ExosomeExosomes derived from metastatic breast cancer cells drive signaling pathways for PI3K/AKT mitogen-activated protein kinase (MAPK), which stimulate migration and invasion of cancer cells by degrading ECM components . Moreovevia modulating Wnt/β-catenin and ERK signaling pathways promote EMT , notably in response to therapy . These cAlterations in cell metabolism are one of the hallmarks of cancer. Exosomes have long been thought to be essential mediators in cancer progression, regulating extracellular communication with cancer cells . It is wCancer-Associated Fibroblasts (CAFs), a bulk of TME cell types, are characterized by the conversion to a synthetic phenotype which could be ascertained by the expression of markers such as α-smooth muscle actin (α-SMA), fibroblast-specific protein-1 (FSP1/S100A4), and fibroblast activation protein (FAP) . Cancer via the TCA cycle and OXPHOS. This phenomenon is known as the “Reverse Warburg Effect” , and other therapeutic compounds  Vega et. ExosomeThe modified exosomes have significant anti-neoplastic effects in pancreatic adenocarcinoma cells of humans . ExosomeExosomes might be used to deliver anticancer proteins to cancer cells with pinpoint accuracy. TNF-related apoptosis-inducing ligand (TRAIL) activates death receptors in cancer cells, causing them to perish Because clinical use of nucleic acid medications is limited due to insufficient delivery methods, exosomes may be a good vehicle for delivering therapeutic nucleic acids. RNAs (siRNA and miRNA) have been delivered to tumor cells using exosomes. Exosomes derived from adipose stem cells were utilized to provide miR-122, making hepatocellular carcinoma chemoresistant . ExosomeClinically, nanotechnology-based drug delivery systems are among the most promising tools to achieve this goal. Compared with liposome nanomaterials, metal nanomaterials, and polymer nanomaterials, exosomes as carriers can overcome the shortcomings of poor bioavailability and reduce non-targeted cytotoxicity and immunogenicity . And exoin vitro  is considered to be achievable. Existing results have shown that exosomal Wnt from fibroblasts could induce dedifferentiation of cancer cells to promote chemotherapy resistance in CRC, suggesting that interference with exosomal Wnt signaling could help improve chemosensitivity and treatment window. Exosomes are projected to be beneficial in treating cancer as a whole, with uses in biomarker detection, tumor immunology, and drug delivery. They might potentially alter medicine as a significant participant in theranostic oncology.+ effector T cells in the circulation system of cancer patients were induced apoptosis by TEXs, which was one of many immunoinhibitory mechanisms of TEXs and suppressed the patient’s general immune system. As a carrier for delivery products, exosomes can initiate antitumor immune responses with significant therapeutic effects on tumor progression , and enhance immune-induced apoptosis  as India. Exosomes could have prospected as plausible biomarkers of aggressive cancers as they play an intrinsic role in the etiopathogenesis as well as clinical prognosis. Based upon the origin of the exosomes, we hypothesize that it could be prospected as an attractive theranostic target to create novel immune-surveillance strategies to enable large-scale screening of aggressive cancers at the community level, taking into account the wide variation in the host genetics along with socio-cultural norms. This will help predict the emergent cancer hotspots, which could be seamlessly visualized on digital platforms such as Google Earth. Such a strategy will enable administrators/policy decision-makers at local, regional, national, and global levels to seamlessly allocate the clinical resources to mitigate the lurking epidemic of various cancer at the community level. This kind of community-driven strategy for cancer screening would form the rationale for developing comprehensive cancer management/mitigation strategies which will use computational platforms to develop fruganomic, cutting-edge, niche-specific cancer control strategies intrinsic to the health status of the populace subsisting in a particular area.