Extrachromosomal DNA (ecDNA) is a cancer-specific circular DNA molecule that is derived from chromosomes. In contrast with linear chromosomes, ecDNA exhibits a unique structure that can be representative of high chromosome accessibility, contributing to hyperactivated proto-oncogenes and malignant behaviours. Meanwhile, nonchromosomal inheritance and recurrent mutations of ecDNA fuel tumour heterogeneity and evolution. Recent studies have demonstrated that ecDNA drives tumorigenesis and progression and is related to poor clinical outcomes and drug resistance across widespread cancers. Although ecDNA was first observed in 1965, with technological advancements, its critical functions in tumorigenesis are currently coming forth. In this review, we summarize the current understanding of the origin, biogenesis process, discovery history, molecular mechanisms, and physiological functions of ecDNAs in cancer. Additionally, we highlight the effective research methods to study ecDNA and offer novel insights for ecDNA-directed therapies. Eukaryotic genomic DNA is packaged into linear chromosomes. For various reasons, some genomic DNA can separate from the chromosomes and form particles of different sizes. Biophysical methods and DNA sequencing confirmed that these particles were circular, and thus, they were named extrachromosomal circular DNA (eccDNA). They can encode regulatory elements  and genes. To date, eccDNA particles have been found in a variety of eukaryotes, including yeast Extrachromosomal DNA is also known as double minutes (DMs) Figure 1 shows the timetable for several milestones of ecDNA.Before engaging in ecDNA research, it is necessary to understand some of the pioneering events of ecDNA. In 1965, ecDNAs were found in metaphase neuroblastoma as small chromatin bodies From the late 1970s to the early 1980s, scientists conducted a landmark molecular-level study. ALT et al. first found that an unstable copy number increase in the dihydrofolate reductase gene (DHFR) resulted in resistance to methotrexate in mouse lymphoma cells, which was associated with ecDNAMore than 20 years later, ecDNA has returned to the public view, and studies are gradually emerging. In 2011, the formation of ecDNA during chromothripsis was thoroughly elucidated via next-generation sequencing technology In the last two years, increasing breakthroughs have been achieved in ecDNA. Recent studies have shown that ecDNA is capable of driving somatic rearrangement in neuroblastoma, and thus is a key genomic feature of cancer The biogenesis of ecDNA is not exactly the same as that of eccDNA. There are many pathways of ecDNA biogenesis, and each pathway is accompanied by DNA damage and loss of tumour suppressor genes.Figure A, topAs the most common mechanism for ecDNA biogenesis, chromothripsis occurs when a chromosome suffers catastrophic DNA damage and breaks into several DNA fragments of varying sizes. These DNA fragments then rearrange. The above process acts as a strong driver of tumorigenesisFigure A, bottom. The process begins with the chromothripsis of one or more chromosomes. Then, these fragments reassemble into circular DNA structures (II). Third, these circular DNAs without centromeres undergo sequence recombination to form large circular DNA fragments or linear fragments (Ⅲ). On the other hand, these circular DNAs with centromeres form linear fragments with centromeres through the circular breakage-fusion-bridge (BFB) cycles (IV), which is a BFB cycles variant that acts on circular ecDNA . Eventually, these fragments participate in the next stage of evolution and form three products (Ⅴ), namely, double minutes (DMs), which retain the circular structure; homogeneous staining regions (HSRs), which are generated by the integration of the circular sequence into the chromosome and neochromosomes (NC), which refers to the aberrant chromosome In 2021, Rosswog et al. proposed a new amplification pattern that is completely different from other amplification patterns, namely, “seismic amplification”. More interestingly, they found that this amplification pattern is a dynamic evolution process and is closely related to chromothripsis Recurrently chromothripsis is the driving factor leading to drug resistance of tumour, but DNA repair pathway is necessary for recombination of chromothripsis. Therefore, we suspect that the development of combination therapy targeting these processes may significantly reduce tumour resistance and improve patient outcomes.Figure BThe breakage-fusion-bridge (BFB) cycles, a mechanism for ecDNA biogenesis Studies of ecDNAs and their chromosomal origin have revealed that ecDNAs might originate from slight DNA damage. In the 1980s, because of the lack of available detection methods, it was difficult to achieve comprehensive genomic profiling. Therefore, Wahl et al. utilized Southern blotting to explore the dynamics of episomes  in an analogue system. They demonstrated that the biogenesis of ecDNA was related to the deletion of chromosome fragments Figure A. (2) The sequence of double-stranded DNA is cut off between two replication forks and then forms ecDNA. The scars left on the replication fork can be healed by homologous replication Figure BMore importantly, it is also possible that the origin chromosome has no missing gene locus matching ecDNA fragments. The above findings suggest that ecDNA biogenesis may occur in two aspects: (1) EcDNA consists of fragments that have fallen off from identical or dissimilar chromosomes. The original chromosome will exhibit a scar Figure CThrough the analysis of the ecDNA breakpoint sequence, a possible mechanism related to ecDNA biogenesis, replication fork stalling and template switching was clarified New research has begun to focus on the genetic background of ecDNA. It is well known that the loss of tumour suppressor genes is a manifestation of genomic instability. Smolen et al. found that deletion of tumour suppressor genes, including Trp53 and Brca1, plays an integral role in the amplification of the ecDNA-containing oncogene MET (ecMET) in mouse breast cancer cells Although ecDNA has been formed, it keeps evolving. Previous studies have suggested that the larger ecDNA is formed by episomes expanding in cells th and early 21st century, the ultrastructure of ecDNA was further explored by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Nevertheless, owing to the limited resolution of SEM and AFM, the observed images were not consistent with the true structure of circular ecDNA To date, research on the structure of ecDNA remains the focus of much attention. Previous ultrastructural analyses have elucidated some fundamental characteristics of ecDNA by transmission electron microscopy (TEM). First, ecDNA consists of chromatin harbouring nucleosomes, which are then woven into a typical chromosomal structure. Second, studies confirmed that there are no free sequences of ecDNA, and that its structure seems to be circular In 2019, a study confirmed that ecDNA exhibits a circular structure by DNA sequencing, ultrastructural imaging and long-range optical mapping. First, WGS with amplicon architecture analysis expounded the circular structure developed by some DNA fragments with breakpoint religations between them. Second, long-range optical mapping clarified a consecutive contig that steps over all breakpoints of the ecDNA and confirmed the circular structure of ecDNA. Third, these ultrastructure images, including SEM, TEM and three-dimensional structured illumination microscopy, indicate that the ecDNA is unequivocally circular Cancer genomes feature a mass of somatic mutations that qualify cancer cells with survival advantages. Earlier studies have reported some clustered somatic mutations, such as clustered single-base substitutions, diffuse hypermutation (termed omikli), and longer strand-coordinated events (termed kataegis). Recently, Bergstrom et al. observed multiple kataegis hypermutation on ecDNA (termed kyklonic hypermutation) Figure A. Importantly, recurrent kyklonic hypermutation has been observed across widespread cancers, including glioblastomas, lung cancers and other malignant cancers ecDNA containing cancer-related genes often suffers kyklonic hypermutation, which fuels the evolution of ecDNA. A higher frequency of kyklonic hypermutation in ecDNAs loaded with cancer-related genes has been observed Additionally, they also revealed that the kyklonic hypermutation within ecDNAs was dominated by APOBEC3 Figure B. Given that the circular topological structure endows ecDNAs with higher chromatin accessibility, ecDNA boasts higher transcriptional activity. Consistently, recent studies have reported that even chromosomal DNA and ecDNA have similar copy numbers, ecDNA exhibits a huge advantage in transcribing oncogenes Through the analysis of allele-specific RNA sequencing, it was found that ecDNA can be used as a template for gene transcription The information encoded in DNA is usually determined by its physical appearance. As long as the DNA forms a circular structure, the fragments of ecDNA will develop a novel chromatin domain that is different from their linear chromosome structure. Interestingly, because DNA forms a circular conformation, it is possible to bring distant DNA elements (enhancers) nearby, forming a new cis-regulatory configuration that is impossible for chromosomal DNA Figure C, top. As long as the enhancers and the oncogene coexist in an ecDNA circular domain, the enhancers can cross the insulator and participate in the transcriptional regulation of the oncogene Figure C, bottom. It has been found that the oncogene EGFR often co-amplifies with upstream enhancers and forms ecDNA in glioblastoma, generating new enhancer-oncogene contacts and promoting tumour progression Enhancer hijacking that occurs on ecDNA typically exhibits two patterns: the local enhancer hijacking model and the distal enhancer hijacking model. In the local enhancer hijacking model, the ecDNA circularizes and hijacks the enhancers at the distal end of the oncogene to bring them into the vicinity. Insulators insulate enhancers from oncogenes, which can prevent enhancers from participating in oncogene regulation in the chromosomal DNA Figure D. These DNA segments containing enhancers may originate from identical or dissimilar chromosomes. Thus, distal enhancer hijacking creates a unique domain on ecDNA that complicates oncogene regulatory mechanisms on ecDNA. EcDNA-mediated remote enhancer hijacking is prevalent in neuroblastoma, which often leads to a poor prognosis The distal enhancer hijacking refers to the process in which distant enhancers and oncogene fragments join together to form ecDNA loops Additionally, we believe that ecDNA could employ epigenomic factors to gain survival advantages. EGFR ecDNA formation relies on H3K9 methylation DNA is wrapped tightly around histones and packed into a linear chromosomal structure. Different chromosomes occupy specific spaces within the nucleus, which forms chromosomal domains. This spatial structural domain is significant for the function and physical stability of chromosomes, and can also limit the interactions of chromosomes Figure E. Interactions between ecDNA and chromosomal DNA appear to be genome-wide. Moreover, up to a few hundred trans interaction sites, where ecDNA interacts with chromosomal DNA, were illuminated by RNA polymerase II ChIA-PET , which further confirmed that their interactions contribute to higher transcriptional activity. Furthermore, ecDNA contains a large number of enhancers, and these enhancers often interact with chromosomes. From what has been discussed above, ecDNA is involved in regulating the expression of chromosomal genes, especially oncogenes, owing to the characteristic of its mobile enhancer A later study regarding interactions between ecDNA and chromosomal DNA indicated that the function of ecDNA equates to a movable trans-acting element Figure F. Recent studies have shown that ecDNA tends to physically aggregate, and ecDNA elements can gather to form ecDNA hubs Figure F, which can promote the expression of oncogenes. For instance, ecDNAs containing MYC-PVT1 without enhancers can hijack the enhancers from other ecDNAs within the ecDNA hub to enhance the expression of the fusion gene Due to the high level of amplification and mobility of ecDNA, the interaction between ecDNA elements is expected Figure A, left, and damage events of existing ecDNA can also lead to the deletion of a certain sequence Figure A, rightNow, the concepts towards the instability of ecDNA can be extended to the changes in physical conformation and spatial dynamics, where ecDNA may alter their sequence and localization in the nucleus Figure B, top, and can also act as a hijacked enhancer to regulate TERT expression in neuroblastoma Figure B, bottomEarly observations of Southern blotting and contemporary sequencing techniques combined with bioinformatics analysis found that some ecDNAs are able to aggregate and subsequently reintegrate into chromosomal DNA Figure CInterestingly, since the fragments forming ecDNA may come from different chromosomes, ecDNA biogenesis is also accompanied by gene fusion  Figure A-G.ecDNA has been observed in a variety of cancers and plays a key role in tumour progression. In this section, we present recent reports exploring our understanding of ecDNA's biochemical functionsFigure A68. deCarvalho et al. demonstrated that both ecDNA-mediated oncogene amplification and somatic single-nucleotide variants are involved in the dynamic evolution of glioblastoma ecDNA initiates a great number of carcinogenic amplifications and mutations and has been identified in 10-40% of glioblastomasThe amplification of the epidermal growth factor receptor (EGFR) gene through double minutes is frequently observed in glioblastoma Figure A. Thus, oncogenes amplified on ecDNA might also serve as predictive biomarkers for therapies In glioblastoma, EGFR is frequently mutated, forming the oncogenic variant EGFRvIII. EGFRvIII prompts tumour growth but makes glioblastoma cells more sensitive to EGFR tyrosine kinase inhibitors (TKIs) Subsequently, a new type of mutation in cancer, termed amplification-linked extrachromosomal mutations (ALEMs), was proposed. Oncogenic focal amplification of some oncogenes (such as EGFR and PDGFRA) on ecDNA may increase the chance of functional mutations. Amplification-linked extrachromosomal mutations (ALEMs) are common in glioblastoma and low-grade gliomas, as well as other tumours. The ecDNA-mediated ALEMs explain the amplification of mutated oncogenes, including EGFR and PDGFRA, in glioblastoma Figure B. Moreover, they explored the correlations between the focal amplifications (including ecDNA-derived circular amplicons) and prognosis based on an immunohistochemistry analysis from 1,688 gastric cardia adenocarcinoma patients.Oncogenic focal amplification plays a pivotal role in the progression of gastric cardia adenocarcinoma and is associated with poor prognosis The results show that ERBB2-positive patients have worse prognosis than ERBB2-negative patients when their survival time is less than two years. Notably, if the patients' survival time is longer than two years, the tendency could be completely reversed. Therefore, ecDNA-derived ERBB2 focal amplifications might serve as a favourable prognostic biomarker in gastric cardia adenocarcinoma patients As mentioned above, further analysis of ecDNA in GCA progression is quite meaningful and may provide several more sensitive biomarkers and effective targets for therapies.Figure C. Additionally, withdrawing MTX treatment decreased ecDNA-mediated DHFR amplification in MTX-resistant cells. The loss of the DHFR amplicon in MTX-resistant cells can suppress their capacity to generate resistance. As expected, when these MTX-resistant cells that lost the DHFR amplicon were re-exposed to MTX, the cells may become responsive to the second round of MTX treatment. These observations provide a promising treatment strategy for overcoming drug resistance induced by ecDNA-mediated amplification As one of the oncogenic genomic features, gene amplification can markedly prompt tumour evolution and drug resistance Figure DJin et al. found that noncoding regions on ecDNAs perform a considerable function in regulating gene expression. They discovered several matrix attachment regions (MARs) within an ecDNA derived from UACC-1598 cell line using sequence analysis and bioinformatics analysis. Moreover, they have identified the interaction between the MARs and the nuclear matrix, which results in a significant enhancement of gene expression. Transfecting the MAR construct into 293 T cells could also enhance the expression of oncogenes located near the MARs, including MYCN and EIF5A2 Raymond et al. performed a clinical trial to evaluate the efficacy of hydroxyurea on inhibiting double minutes in cancer cells from patients with ovarian cancer. They demonstrated that low-dose hydroxyurea could decrease the number of double minutes in cancer cells Although the role of ecDNA in ovarian cancer is not thoroughly understood, further research is warranted. On the one hand, noncoding regions of ecDNA can cause increased oncogene expression, which may provide a new target for ovarian cancer treatment Figure EPrevious reports have theorized that hybrid human-virus ecDNA formation could be a potential mechanism for increased expression of the HPV oncogenes E6 and E7 Later, Deshpande et al. demonstrated that the HPV-mediated oropharyngeal cancer cell line UPCI: SCC090 contains hybrid human-viral ecDNA Human-viral hybrid ecDNA is an exclusive feature of HPVOPC. In this way, the hybrid ecDNA boasts an uneven inheritance, thereby driving rapid tumour evolution even in the face of targeted therapies. Thus, the characterization of ecDNA in oropharyngeal cancers will provide some prognostic biomarkers for clinical treatment. Additionally, clarifying the role of ecDNA in HPVOPC will contribute to the development of HPVOPC therapy.Figure F. Moreover, they demonstrated that RAB3B protein could induce a cisplatin resistance phenotype in HSCC by promoting autophagy. However, they did not verify that the sequences of the RAB3B genes on ecDNA can be directly transcribed. ecDNA-mediated drug resistance-related gene amplification might explain cisplatin resistance in hypopharyngeal squamous cell carcinoma (HSCC) Cisplatin resistance leads to the malignant progression of hypopharyngeal squamous cell carcinoma (HSCC). Recently, Lin et al. found that the gene encoding RAB3B might be amplified on ecDNA Figure GThe first appearance of ecDNA was observed in the metaphase of neuroblastoma cells in 1965 Figure GRecently, it has been reported that ecDNA can drive oncogenic genome rearrangement in neuroblastoma The abovementioned molecular signatures are being evaluated to better understand the disease, which would help identify novel targets and improve the treatment outcomes of patients with neuroblastoma.With the rapid development of the ecDNA field, robust tools are continuously emerging. Here, we will present current effective tools for ecDNA research.Sequencing and analysis tools can be attractive solutions to assessing some basic properties of ecDNA from publicly accessible cancer genome databases and patient-derived tumour tissue. Here, we present some effective sequencing-based approaches for ecDNA.Figure A89. Modern ecDNA isolation involves two new steps: 1) column- or magnetic bead-based methods to isolate high-molecular-weight DNA and 2) removing linear DNA by exonuclease Figure B90. Additionally, the enrichment protocols require rolling-circle amplification of ecDNA before sequencing Figure C. In neuroblastoma, circle-seq has revealed the landscape of MYCN amplicons, which are amplified on ecDNA Circle-seq is a specialized sequencing method for ecDNA https://github.com/crimBubble/ECCsplorer). Following circle-seq, ECCsplorer enables convenient and efficient discovery of potential ecDNA Figure D91.Recently, Mann et al. developed a bioinformatics pipeline named ECCsplorer  can provide access to all information about the genome in an organism. Searching for discordant reads is a viable method for identifying ecDNA. In general, 10× WGS coverage is adequate to detect ecDNA Figure F28. The combination of WGS and optical mapping, in conjunction with AmpliconReconstructor, a recently developed algorithm, is proven to be an extremely accurate and economical technique for resolving complicated amplicon architectures The main problem with WGS, however, is that its read length is relatively short (100 to 200 bp); it cannot identify the complex structural rearrangements of amplicons as large as ecDNA, which is often between 1-3 megabases in length. Technologies such as nanopore long-read sequencing and optical mapping are proven methods to resolve the ecDNA structure and can mitigate this limitation  Figure G94. In addition, sequenced genomic fragments usually do not contain exclusive junction sequences, thus making it impossible to differentiate the genomic segments from different ecDNA. To improve our efficiency for identifying ecDNA by sequencing, a side-by-side comparison of WGS and ATAC-seq followed by their respective software analysis is necessary.Other sequencing and analysis tools, such as ATAC-seq with Circle-finder software, could also identify ecDNA from cancer tissueshttp://circlebase.maolab.org), that integrates and analyses human ecDNAs from accessible public data. Moreover, it can predict the regulatory networks among ecDNAs by integrating and analysing relevant databases. CircleBase boasts several advantages. (i) CircleBase enables visualization of the functions and annotations of ecDNAs; (ii) CircleBase is equipped with a ranking system (based on the Gaussian distribution model) for ecDNA and (iii) provides overall ecDNA annotations Figure H95. Thus, CircleBase is a robust tool for explaining the functions and mechanisms of ecDNA and will facilitate the exploration of tumour heterogeneity and genome diversity.Zhao et al. have developed a new platform, CircleBase . Although it provides very high precision, it achieves a slightly lower sensitivity than visual counting and thus quantifies less ecDNA, especially ecDNA particles near chromosomes Figure J. With the help of ecTag, the spatial and temporal dynamics of ecDNA can be effectively captured. Moreover, the hypothesis that uneven segregation of ecDNA could contribute to intratumoral heterogeneity has been further confirmed by ecTag Advances in biological techniques have also greatly improved the detection of ecDNA. Recently, Yi et al. developed a CRISPR-based ecDNA tracking system called 'ecTag'. Leveraging DNA junction sites, ecTag can label ecDNA with multiple fluorescent molecules in living cells Nevertheless, imaging-based technology has some limitations. Occasionally, it is not feasible to prepare metaphase cells. FISH cannot be performed without sequence information for the ecDNA. In addition, the detection throughput of the imaging approach is low. Consequently, well-suited, sensitive, and high-throughput tools are needed.Figure A. Additionally, we believe that the ecTag might uncover the mechanisms contributing to ecDNA elimination by tracing the spatiotemporal dynamics of ecDNA moving into micronuclei.Cancer cells gain survival advantages by continually altering their genomes. Oncogene amplification is a classic form of genome alterations. Cancer cells are addicted to ecDNA because ecDNA is a carrier that maintains oncogene amplification Figure B.Tumour heterogeneity is an important cause of drug resistance, and the increased frequency of ecDNA reintegration may reduce heterogeneity among cancer cells. Recently, PARP has been shown to reduce the frequency of ecDNA reintegration and thus may be a candidate therapeutic target Figure C.Reducing the risk of ecDNA biogenesis might serve as a promising treatment for cancer patients. Nonhomologous end joining during DNA damage repair is involved in ecDNA generation, and DNA-dependent protein kinase inhibitors can interfere with this process. The emergence of ecDNA is significantly reduced after treatment with the drug Figure D. If we want to treat cancer by interfering with the formation of ecDNA hubs, there are still some aspects that need to be elucidated. For example, are the segregation of ecDNA in the form of singletons or smaller hubs? Whether the composition of ecDNA hubs changes with cell passage? Whether the spatial distribution of ecDNA hubs in the nucleus is random or directed? Are there differences in the composition of ecDNA hubs in different cancer species?The spatially abnormal distribution of ecDNA, which eventually forms ecDNA hubs, can cause trans-interactions between ecDNA and between ecDNA and chromosomal DNA. Therefore, the spatially abnormal distribution of ecDNA may represent a treatment-related vulnerability. Proteins involved in the formation of ecDNA hubs have been considered as emerging potential therapeutic targets Given the large number of new research results on the occurrence, maintenance and function of ecDNA, it is feasible to develop related drugs targeting ecDNA. In the future, therapeutic targets of ecDNA may develop as a new approach to conquer tumours.ecDNAs are circular DNA that contain oncogenes and are responsible for tumorigenesis. Numerous mechanisms have been proposed to clarify the genesis of ecDNA, including chromothripsis, slight DNA damage and relegation, fork stalling and template switching, and genome instability. However, further studies are required to reveal the mechanisms by which these ecDNAs are formed. ecDNAs exhibit unique structure and function which enable them to accelerate the evolution of tumours. ecDNAs contribute to oncogene overexpression and somatic rearrangement, ultimately causing cancer progression and tumour heterogeneity. These processes eventually lead to cancer cells becoming resistant to various stresses and therapies. A series of pioneering tools for ecDNA research have been developed in recent years. For example, the development of “ecTag” has removed the barriers in exploring the temporal dimension of ecDNA. Further understanding of ecDNA requires continuous exploration and advancement of technology.The field of ecDNA is rapidly advancing. Currently, there are still some key areas that must be addressed. The first is clarifying the basic biology of ecDNA. It is necessary to detail the mechanisms of chromosomal instability in ecDNA formation and further explore the roles of NHEJ pathways and double-strand breaks in this process. Furthermore, the influence of ecDNA reintegration on genome rearrangement and its relocation as HSR is still not fully understood. It has been reported that HSR formation is associated with drug resistance, but the detailed mechanisms remain largely unknown.Second, ecDNA is correlated with tumour evolution. In the future, unremitting explorations will be necessary to better understand the molecular pathogenesis mechanisms and function of ecDNA in tumours. Moreover, a recent study demonstrated that ecDNA can activate innate immune signalling pathways such as cGAS-STING. Thus, the role of ecDNA in the tumour immune microenvironment deserves more attention.Third, as we deepen our understanding of ecDNA, we will be able to overcome ecDNA-associated clinical pathogenesis. Due to their relative stability and association with human diseases, ecDNA molecules may serve as sensitive biomarkers, which will enable early diagnosis. Moreover, as a predictive biomarker, ecDNA might provide patients with instructive guidelines for the choice of chemotherapy, targeted therapy, or immunotherapy, thereby realizing individualized precision medicine. On the other hand, given the important role of ecDNA in cancer progression, developing drugs targeting ecDNA is promising for the future.Additionally, robust tools are needed. Improving current enrichment and sequencing methods will make the process of extracting circular amplicons more effective. The development of computational or analytical tools such as long-read and single-cell sequencing will facilitate the construction of circular amplicons. As the tools advance, the chromatin structural features of ecDNA will be systematically explored; cancer-associated sequence characteristics of ecDNA will be precisely identified; and a thorough understanding of ecDNA will be gained.


When the human mind wanders, it engages in episodes during which attention is focused on self-generated thoughts rather than on external task demands. Although the sustained attention to response task is commonly used to examine relationships between mind wandering and executive functions, limited executive resources are required for optimal task performance. In the current study, we aimed to investigate the relationship between mind wandering and executive functions more closely by employing a recently developed finger-tapping task to monitor fluctuations in attention and executive control through task performance and periodical experience sampling during concurrent functional magnetic resonance imaging (fMRI) and pupillometry. Our results show that mind wandering was preceded by increases in finger-tapping variability, which was correlated with activity in dorsal and ventral attention networks. The entropy of random finger-tapping sequences was related to activity in frontoparietal regions associated with executive control, demonstrating the suitability of this paradigm for studying executive functioning. The neural correlates of behavioral performance, pupillary dynamics, and self-reported attentional state diverged, thus indicating a dissociation between direct and indirect markers of mind wandering. Together, the investigation of these relationships at both the behavioral and neural level provided novel insights into the identification of underlying mechanisms of mind wandering. The phenomenon of mind wandering in humans can be described as the spontaneous stream of consciousness that comprises thoughts, emotions, and memories  that areNotwithstanding the diversity of contexts in which mind wandering has been previously investigated, researchers continue in their pursuit to further unravel its underlying neural mechanisms and its effect on other cognitive processes and behavior. In particular, although there is little debate regarding the involvement of executive functions in mind wandering in general, there is no consensus on exactly how mind wandering interacts with executive control systems and whether it is better characterized as executive function use  or as thThe majority of mind wandering research reports data from self-reports through periodical thought probing and performance errors, usually during a sustained attention to response Task SART; . HoweverBesides monitoring executive function use, findings from several studies suggest trial-to-trial response time variability as a promising and sensitive marker for fluctuations in attentional focus , especiaSimilarly, several studies have attempted to identify psychophysiological markers reliably related to an individual’s attentional state. In particular, a growing body of evidence suggests that spontaneous changes in pupil size are linked to dynamic fluctuations between internal versus external attention and awareness . ChangesIn summary, the DMN, attention and executive control networks, and the LC/NE-system are all implicated in mind wandering but empirical evidence into how these neural systems interact to give rise to mind wandering is at present incomplete. Building on previous findings, we aimed to address this by employing an fMRI version of the FT-RSGT that combines experience sampling with objectively defined measures interpreted as indirect makers for changes in ongoing attentional state, including sensitive behavioral indices and pupillometric measures. Following Additionally, we explored the pattern of neural activation in relation to both direct and indirect markers of mind wandering by directly assessing the patterns of network-wide activity in the periods preceding experience sampling probes as well as the brain regions that correlated with behavioral performance. In line with a previous finger-tapping study, we expected to observe recruitment of ventral and dorsal attention networks and cerebellum when variability in finger-tapping was high and DMN activation when finger-tapping was more stabilized . The degSD = 7.2 years) who were recruited from the Amsterdam ultra-high field adult lifespan database  every 750 ms. Previous experiments determined that this pace is optimal for engaging in generating random sequences compared to slower and faster metronomes . During m that are similar remain similar for subsequences augmented by one position  measured in the FT-RSGT correlated with the entropy measure in a keyboard version of the original RNGT, validating its use as an index for executive control (Z-scored) across subjects . The choice of the 25-tap window was decided a priori based on the assumption that mind wandering occurs in slowly fluctuating episodes spanning multiple seconds as well as for ensuring that sufficient data was gathered for reliable calculations of BV and AE. Additionally, previous experiments revealed that BV and AE based on this window size had the strongest relationship with self-reported mind wandering compared to shorter windows  was calculated as the standard deviation of the intertap intervals (ITIs) of the 25 finger-taps preceding each thought probe. No filtering or preprocessing was performed so that missing or double taps per trial were included in the calculation. The raw standard deviations were log-transformed to approximate a normal distribution. Second, the degree of randomness in the self-generated sequence of left and right finger-taps was measured with the approximate entropy (AE) metric . As the  control . AE was  windows .The pupil area of the left eye was concurrently recorded during the fMRI session at a sampling rate of 1000 Hz with the Eyelink 1000 Plus tracking system (SR Research). The pypillometry package  was usedh = net-n/tmax, where n = 10 and tmax = 900; b coefficients. However, predictor multicollinearity was observed as stimulus and tap onsets occurred close in time. Therefore, the final single-trial phasic pupil responses were calculated as the sum of b coefficients from all events located within the 200-ms window before and after each stimulus onset. Finally, single-trial tonic and phasic pupil responses were standardized (Z-scored) within subjects to remove incidental differences in absolute pupil size across subjects. More details on the deconvolution-based pupil analysis are described by Selecting specific windows for extracting the mean pupil signal or peak amplitude is complicated in fast-paced task designs due to the build-up of evoked pupil responses that resemble increases in baseline pupil size and therefore contaminate the baseline estimates of subsequent trials. Therefore, to produce more valid estimates of single-trial tonic (baseline) and phasic (evoked) pupillary dynamics, a recently developed deconvolution-based approach was applied . First, T1-weighted (T1w) images were acquired with a turbo field-echo (TFE) sequence in 257 sagittal slices . Whole-brain functional images were acquired in a single fMRI run with single-shot fast field-echo (FFE) echo-planar imaging (EPI), collecting 56 transverse slices per volume with 0.2 mm slice gap . An additional EPI field map with opposite phase-encoding direction was acquired to measure and correct for field distortions.Participants were scanned with a 3Tesla Philips Achieva MRI system with a 32-channel head coil. T1w) images were corrected for intensity non-uniformity with N4BiasFieldCorrection  or negative (ER−) effect. The ER is calculated as the ratio between the probability of the effect being positive divided by the inverse probability of the effect being zero or negative (ER+) or the inverse of that ratio (ER−) and can be interpreted as an odds-ratio. We consider an effect as reliable when the area under the marginal posterior distribution that is larger than zero (for ER+) or smaller than zero (for ER−) is >0.95 (corresponding to an ER of 19 and the 95% HDI excluding zero). The models were fitted with four Hamiltonian Monte Carlo (HMC) chains, each with 1000 warm-up and 4000 post warm-up samples.The relationships between self-reported mind wandering, behavioral performance, and pupillary dynamics were assessed with regression models using the thought probe responses from the experimental blocks (random task) of the FT-RSGT as dependent variable. Treating the ordinal probe responses as continuous introduces a range of statistical problems, including poor model fitting, low power, increasing the risk for type I and II errors, and spurious interaction effects . To circ+) and for AE to be smaller than zero (ER−). Similarly, the effects of tonic and phasic pupillary dynamics as well as the tonic × phasic interaction were assessed in addition to time in a second regression model using the 18 random blocks from the 21 subjects with complete pupil datasets. For every thought probe, the extracted single-trial tonic and phasic features were averaged across the preceding 25 trials (18.75 s), ignoring trials with more than 40% missing pupil data. This criterion resulted in exclusion of six thought probes, therefore the model was fitted on 372 thought probe responses in total. The evidence for effects larger than zero (ER+) for tonic pupil size and the tonic phasic interaction and smaller than zero (ER−) for phasic pupil responses was evaluated.In the first probit model, the effects of time (probe number), BV, AE, and the BV × AE interaction on 486 thought probe responses (27 subjects × 18 blocks) were modeled. In accordance with our hypotheses, for time, BV, and BV × AE, we evaluated the evidence for the effect to be larger than zero  were fitted to the fMRIPrep-preprocessed time series using FSL FEAT  to exploFirst, we explored the hypothesis that random-sequence generation recruits more widespread executive and attentional networks compared to alternating finger-tapping. In addition to the task-related and nuisance regressors, the occurrence of random and alternating blocks was modeled by two boxcar functions convolved with a double-gamma HRF. In a second model, patterns of brain activity associated with episodes of mind wandering compared to on-task thoughts were investigated by convolving boxcar functions that modeled the 10s intervals preceding off-task versus on-task thought probes . To accoThird, regressors for task performance were modeled to evaluate brain activity corresponding to increases and decreases in BV and AE. Starting at the 25th trial (metronome onset) per block, BV and AE (random blocks only) were calculated for every single trial based on the preceding 25 finger-taps. To create parametric regressors, single-trial BV and AE were transformed and standardized across subjects before nearest-neighbor interpolation and resampling to the resolution of the fMRI timeseries . The useFor every resulting group-level statistical map we calculated the overlap with the 7- network parcellation , the HarSD = 1.46, median = 3) on the six-point Likert-scale, demonstrating that participants reported that their thoughts were more often focused on performing the task rather than being engaged in mind wandering. Indeed, five subjects never reported that their thoughts were in any of the three off-task categories. There was no significant difference in mind wandering propensity between the random  and alternating task conditions  = −0.53, P = 0.602). Mean behavioral variability was, however, significantly higher during random  compared to alternating finger-tapping  = 3.30, P < 0.01) suggesting that the additional task of generating random sequences interfered with maintaining synchronized motor responses to the externally cued rhythm.In total, 36% of probe responses were in one of the three off-task categories. The mean probe response given by participants was 2.89  were positive whereas the coefficient for AE  was negative. Thus, mind wandering self-reports were more frequent as the task progressed and were preceded by increases in tapping variability and decreases in self-generated sequence randomness although the HDI of the latter coefficient did not exclude zero. The direction of the BV × AE interaction suggests that the relationship between BV and mind wandering was stronger at high levels of AE and weaker (but still positive) at low levels of AE. However, the HDI of this effect included zero, warranting a cautious interpretation of this result. Since BV was significantly higher in the random task, we assessed whether task condition modulated the relationship between BV and mind wandering. We performed a probit model with a BV × task interaction , which indicated that the observed positive relationship between BV and mind wandering was independent from task condition.The unadjusted Bayesian R2 was 0.53 . The positive effect of time on mind wandering was replicated , tonic pupil size decreased as self-reports of mind wandering increased . However, both effects can be considered inconclusive as the HDIs did not exclude zero. Instead, the tonic × phasic interaction  provided evidence of a negative interaction effect. This finding suggests that the relationship between phasic responses and mind wandering is dependent on fluctuations in tonic pupil size. Specifically, the negative relationship between phasic pupil responses and mind wandering, as would be expected due to perceptual coupling, only exists when tonic pupil size ishigh.For the probit model including pupil regressors, the unadjusted Bayesian When contrasted with random-sequence generation, we observed that alternating finger-tapping was associated with localized activity in the posterior cingulate cortex/precuneus and left angular gyrus, both regions that are core nodes of the DMN. In line with our hypothesis, the generation of random tapping sequences instead revealed widespread recruitment of cortical areas generally attributed to attention and executive control networks, including the superior parietal lobe, intraparietal sulcus, primary motor cortex, and dorsolateral prefrontal cortex of the right hemisphere as well as bilateral anterior insula, bilateral medial supplementary motor areas, and bilateral anterior and posterior parts of the cerebellum . TogetheContrary to previous findings, brain activations directly preceding self-reports of mind wandering when contrasted with on-task reports could not be localized to the known cortical nodes of either the DMN or FPCN/DAN. Instead, we observed clusters of brain activation associated with mind wandering in visual, cerebellar, and subcortical areas that could be distinguished between the two task conditions. Specifically, mind wandering in the alternating task was preceded by activity in the left inferior occipital gyrus, temporal subgyral white matter (not plotted on the surface mesh), and parts of the bilateral anterior and posterior cerebellum, whereas mind wandering during the random task was associated with greater activity in the right striatum . To testNext, we assessed patterns of neural activity corresponding to variability in finger-tapping  and obtaSimilarly, we observed correlated neural activity in the expected frontoparietal regions for increases in the degree of randomness in the tapping-sequence as quantified with AE, namely within the right intraparietal sulcus, right posterior middle frontal gyrus, and left medial supplementary motor area, thus showing a similar pattern of neural network recruitment as observed for the random task condition in general. Decreases in AE, signaling decrements in task performance, were instead associated with the left inferior temporal sulcus (poorly visible on the plotted surface mesh) and the superior and inferior occipital gyri of the left hemisphere C. SimilaPositive correlations with tonic pupil size were observed almost exclusively in subcortical and cerebellar areas, including the thalamus, internal capsule, and intracalcarine cortex of both hemispheres as well as the right hippocampus. In addition, a large brainstem cluster covered the locus coeruleus, substantia nigra, and subthalamic and ventral tegmental nuclei. Furthermore, widespread cerebellar activation was observed in the anterior and posterior lobes and dentate nuclei. Interestingly, brain regions that negatively correlated with tonic pupil size largely overlapped with visual and somatomotor cortical network parcellations, including the primary somatosensory cortices, superior frontal gyri, medial primary motor cortices, lateral occipital cortices, superior temporal gyri, hippocampal areas, and cuneal cortices of both hemispheres in addition to the left middle temporal gyrus and right fusiform gyrus . In contTo disentangle the complex interplay between mind wandering, executive functions, and behavior, we employed an fMRI version of a recently developed finger-tapping random-sequence generation task (FT-RSGT). This novel paradigm allows assessment of ongoing fluctuations in task-related and task-unrelated attentional states through self-reports and sensitive indices of task performance at high temporal resolution. Concurrent fMRI and pupillometric measures were used to investigate the neural substrates of direct and indirect markers of mind wandering.Participants completed interleaved blocks of two different finger-tapping tasks, one where they performed alternating sequences and one that required randomized responding, that were otherwise identical in terms of stimulus presentation, task pacing, and thought probe frequency. We therefore hypothesized that the difference in neural recruitment between the two tasks should be mainly reflected in the activation of brain regions associated with executive functioning. Indeed, our results demonstrate greater involvement of attention and executive control networks during the generation of random sequences compared to alternating finger-tapping. Specifically, activation was localized in brain regions previously implicated in random number generation, including the right superior parietal lobe and intraparietal sulcus, primary and supplementary motor areas, right dorsolateral prefrontal cortex (DLPFC), anterior insula, and cerebellum . The DLPIn addition, we observed a similar pattern of frontoparietal cortical activation associated with increases in sequence randomness, including the right intraparietal sulcus, right posterior middle frontal gyrus, and left medial supplementary motor area. This is consistent with an early study employing a finger-tapping task combined with random sequence generation  and suggDecreases in randomness, as would be expected to occur when thoughts drift away from the task as subjects engage in mind wandering, were instead associated with activity in the left inferior temporal sulcus and left superior and inferior occipital gyri. Although these regions are not typically associated with decrements in task performance, a large-scale meta-analysis  reportedWhen executive task demands were low and participants were required to simply produce an alternating finger-tapping sequence, greater activity was observed in the core nodes of the DMN, namely the posterior cingulate cortex/precuneus and left angular gyrus. Given the context-regulation hypothesis, which states that the propensity to mind wander is adaptively adjusted to environmental demands , it is cWe did not observe a difference in mind wandering propensity between the two task conditions, which is surprising given previous studies demonstrating an effect of task difficulty on mind wandering . One posContrary to expectations, the patterns of neural recruitment directly preceding mind wandering self-reports and during periods of increased tapping variability did not converge. However, the observed divergence in our study is less surprising given the results of a previous study employing a continuous performance task , in whicIt should be noted that there exists a large heterogeneity in the design and the direct or indirect measurement of mind wandering across previous studies and different forms of mind wandering or spontaneous thought can be discerned based on their neural correlates . In addiFinally, we investigated the neural correlates of changes in slowly-fluctuating pupil dilations and constrictions as well as changes in the amplitude of evoked transient responses to task-related events as derivatives of tonic and phasic LC/NE dynamics, respectively . In agreInterestingly, occipital activation was frequently either directly or indirectly associated with mind wandering, including when sequence randomness decreased , during tonic  and phasic  pupil constriction, and preceding mind wandering reports in the alternating task , possibly suggesting similar underlying cognitive states. It has been argued that through cortical feedback mechanisms, the occipital cortex may play a role in cognition independent from perceptual input, such as internal visual representations that transpire during mental imagery and mind wandering . TogetheIn summary, we demonstrated that the FT-RSGT relies on the recruitment of attentional and executive control networks, providing evidence for our hypothesis that the generation of random as opposed to alternating finger-tapping sequences requires the use of executive resources. Secondly, we observed positive significant relationships between self-reported episodes of mind wandering and time-on-task as well as behavioral variability, replicating earlier findings and validating the use of this task as an fMRI paradigm . FinallyGrootJM_etal_2021_supplementary_material_bhab494Click here for additional data file.supplementB_bhab494Click here for additional data file.supplementC_bhab494Click here for additional data file.


Regulated cell death (RCD) is central to the development, integrity, and functionality of multicellular organisms. In the last decade, evidence has accumulated that RCD is a universal phenomenon in all life domains. Cyanobacteria are of specific interest due to their importance in aquatic and terrestrial habitats and their role as primary producers in global nutrient cycling. Current knowledge on cyanobacterial RCD is based mainly on biochemical and morphological observations, often by methods directly transferred from vertebrate research and with limited understanding of the molecular genetic basis. However, the metabolism of different cyanobacteria groups relies on photosynthesis and nitrogen fixation, whereas mitochondria are the central executioner of cell death in vertebrates. Moreover, cyanobacteria chosen as biological models in RCD studies are mainly colonial or filamentous multicellular organisms. On the other hand, unicellular cyanobacteria have regulated programs of cellular survival (RCS) such as chlorosis and post-chlorosis resuscitation. The co-existence of different genetically regulated programs in cyanobacterial populations may have been a top engine in life diversification. Development of cyanobacteria-specific methods for identification and characterization of RCD and wider use of single-cell analysis combined with intelligent image-based cell sorting and metagenomics would shed more light on the underlying molecular mechanisms and help us to address the complex colonial interactions during these events. In this review, we focus on the functional implications of RCD in cyanobacterial communities. Genetically regulated cell death (RCD) was first identified in metazoans and later found in prokaryotes and different eukaryotes ,2,3. TheMicrocystis) and/or exhibit multicellularity, with up to six distinct cell types in filamentous cyanobacteria  and [N] levels, phage infection could be activated, and cyanophage would actively utilize host cellular machinery to maintain and expand the phage population [Prochlorococcus, and Bar-Zeev et al. [Trichodesmium. In the latter study, recently fixed nitrogen was sinking into the sediments with the cell for further decomposition by heterotrophic bacteria. Distinct transcriptional states may co-exist during the active viral infection of cyanobacteria: (1) cells resistant to lysis that keep persisting viruses (“persisters”) and could potentially serve as an inoculum for next blooms; (2) metabolically active infected cells responsible for viral release followed by lysis, and (3) a population of cells dying without releasing any viral particles . Similarpulation . At the pulation . Fuchsmapulation  have prov et al.  for a nispeD gene—a homolog found in all marine cyanobacteria—is responsible for polyamine biosynthesis. Along with the psbA gene, also present in the marine phage genome [speD is thought to maintain the activity of the host photosystem II reaction center during phage infection [However, it is unclear what the primary factors that stabilize the host–phage relationship are and whether this relationship is cyclic . Light ae genome , if exprnfection ,148. It Microcystis-associated bacteria and the existence of complementary pathways between Microcystis and associated heterotrophs [Rhodospirillales, Burkholderiales, and Verrucomicrobiales dominated during the rapid decomposition phase, whereas Sphingomonadales, Rhizobiales, and Xanthomonadales dominated during the slow decomposition phase. Changes in a bacterial community are likely involved in the formation and maintenance of Microcystis colonial morphoforms [Microcystis was maintained in the lab culture, colonies contain heterotrophic bacteria [Sphingomonas are associated with Microcystis blooms and actively break down toxins [Microcystis colonies harbor bacterial assemblages that may be conserved across geographically distinct blooms and over different stages of blooms [Microcystis colonies harbor a selected pool of bacteria with a conserved functional potential that may help bloom persistence even under unfavorable environmental conditions. Parasite strategies, such as bacterial regulation of host RCD, can drive the evolution of host–parasite interactions leading to a possible outcome of cell death, dormancy, or infection. If bacteria delay and/or promote Microcystis colony growth and participate in colony decay, they may participate more actively in inducing cell death in the old colonies.Bloom-forming cyanobacteria are closely associated with heterotrophic bacteria ,153,154.rotrophs . The hetrotrophs  observedphoforms . These fphoforms ,158,159.bacteria . Furthern toxins ,162. Sevn toxins . Recent n toxins  confirmef blooms . This fiAnabaena, Aphanizomenon, Cylindrospermopsis, other filamentous taxa, and Microcystis [Asterionella formosa), RCD accelerates the death of infected host cells and the chytrid parasite before it completes its life cycle [In addition to well-studied viruses, many cyanobacteria species are affected by fungal parasites mainly belonging to Chytridiomycota (chytrids) . These mrocystis ,125,167.rocystis . In diatfe cycle . A fungafe cycle  and, thefe cycle  and facife cycle . HoweverEarly examination of phytoplankton automortality characterized by a loss of membrane integrity and degradation of photosynthetic activity, led scientists to supposition of a genetically based process of RCD as a source of phytoplankton mortality . CurrentInitially, the hallmarks of RCD in cyanobacteria were meant to be visualized using a standard for mammalian apoptosis reagents and techniques, such as labeling cell membrane with fluorochrome-conjugated annexin V to verify exposure of phosphatidylserine (PS) ,72,173, . During The cellular ultrastructure of cyanobacteria undergoes significant changes under RCD, and due to the bacterial size, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are the major technologies of choice. A number of studies describing the detailed ultrastructure changes during cell death in cyanobacteria are available ,50,72,73The discovery of RCD-like events in prokaryotes reveals the possibility that widely divergent organisms possess conserved and previously unrecognized RCD pathways . Caspase2O2, salinity changes, and allelopathic stimuli was quantified using a microplate reader or conventional flow cytometer [N-glycanases, inducing autophagy [Most published caspase activity assays in cyanobacteria rely on commercial fluorogenic substrates that produce fluorescence signals upon proteolytic cleavage, indicating the presence of an active caspase . Fluoresytometer ,54,55,73ytometer . Howeverytometer . The panutophagy ,182,183.Recently, the first attempt to compare meta-caspase activity using specific substrates for cyanobacterial caspase-like enzymes and standard substrates for mammalian caspase evaluation was made . The desTargeting intracellular components for degradation is a crucial intracellular process that can counteract the effects of external stress such as invading pathogens . The bioWhen we had already submitted our manuscript, we came upon the interesting recent publication of Li and co-authors 2022) proposing to subdivide RCD cell types in cyanobacteria based on the involvement of caspase homologs . In our 2 proposiSynechococcus and Synechocystis spp.). The key question to be addressed is—are the data obtained with comparative genomics and supporting execution of regulated cell death in multicellular cyanobacteria applicable to unicellular cyanobacteria? We suggest that the enzymes in mechanisms in unicellular cyanobacteria are very different from those in other cyanobacteria groups. In fact, the execution of regulated cellular survival and not a regulated cellular death can be a desired outcome for these bacteria. RCD in cyanobacteria contributes to increased population survival and is an essential part of the ecological process. The multicellularity for many cyanobacterial clades suggests cell-death-related mechanisms of differentiation and protection against infection from viruses, fungi, and other pathogens. In published research, cyanobacteria species used in RCD studies are mainly represented by multicellular filamentous organisms or colonial and unicellular species having reversed from multicellularity in their evolutionary history. On the other side, there are research studies of regulated cellular survival programs in cyanobacteria, mainly using unicellular species (The existing techniques are borrowed from vertebrate RCD research, particularly from mammalian cells equipped with mitochondria—central organelles involved in their process of cell death—and are limited in their capabilities. Further RCD research in cyanobacteria requires the development of proper methods for assessment and wider use of molecular genetics and single-cell analysis in combination with cell sorting based on advanced imaging. This may lead to the discovery of novel RCD molecular pathways and regulatory and inhibitory molecules and contribute to understanding and managing bloom-forming cyanobacteria.


The cancer secretome reflects the assortment of proteins released by cancer cells. Investigating cell secretomes not only provides a deeper knowledge of the healthy and transformed state but also helps in the discovery of novel biomarkers. Secretomes of cancer cells have been studied in the past, however, the secretome contribution of stromal cells needs to be studied. Cancer-associated fibroblasts (CAFs) are one of the predominantly present cell populations in the tumor microenvironment (TME). CAFs play key role in functions associated with matrix deposition and remodeling, reciprocal exchange of nutrients, and molecular interactions and signaling with neighboring cells in the TME. Investigating CAFs secretomes or CAFs-secreted factors would help in identifying novel CAF-specific biomarkers, unique druggable targets, and an improved understanding for personalized cancer diagnosis and prognosis. In this review, we have tried to include all studies available in PubMed with the keywords “CAFs Secretome”. We aim to provide a comprehensive summary of the studies investigating role of the CAF secretome on cancer development, progression, and therapeutic outcome. However, challenges associated with this process have also been addressed in the later sections. We have highlighted the functions and clinical relevance of secretome analysis in stromal CAF-rich cancer types. This review specifically discusses the secretome of stromal CAFs in cancers. A deeper understanding of the components of the CAF secretome and their interactions with cancer cells will help in the identification of personalized biomarkers and a more precise treatment plan. Bacillus subtilis [The term “secretome” was used originally by Tjalsma et al., in the context of proteins secreted by subtilis . In the subtilis . The secsubtilis . A balanPerforming a secretome profile of primary human cells from healthy donors can help in establishing a landscape of a healthy secretome that can be exploited for comprehensive human secretome data in the future. Such a human secretome atlas can be used as a reference for discovery of potential disease-associated biomarkers and eventually novel therapeutic targets . SecretoClinicalTrials.gov. . Thirty-six of studies found are represented in As shown in A deregulation in the constituents of the secretome is associated with development of the cancerous state . The secClinicalTrials.gov. . The studies mentioned below are designed to explore the potential of the secretome in different cancers. When looking for clinical studies investigating cancer secretomes, we found six studies registered in The cancer secretome is also comprised of proteins released from the neighboring cancer-associated stromal cells, such as cancer-associated fibroblasts (CAFs) . One of Carcinoma-associated fibroblasts are activated fibroblasts that represent one of the abundant cell populations present in tumors. CAFs contribute to cancer initiation and progression by delivering oncogenic signals in a paracrine manner . CAFs arAs shown in Cancer-associated fibroblasts have gradually been viewed as key targets for therapeutic advancement in patients with cancer. Exploring CAF-specific features has been advantageous in the hunt for promising cancer biomarkers ,28. SahaAn altered secretome shifts the balance towards substantial deregulation in proteins that promote the oncogenic processes B. The seIdentification and characterization of CAF-derived proteins or the CAF secretome would help in understanding tumor stroma interactions in a way that would enable identification of CAF-specific molecular features that play a crucial role in cancer development. Investigating the CAFs secretome is not only crucial for understanding tumor behavior, but to also discover novel targets for cancer therapies. Investigating the role of the CAF secretome in different cancers is important for gaining novel insights about the tumor–stroma interactions. This section summarizes the data available regarding the CAF-derived secretome. Studies investigating CAFs secretome profiling and analysis were searched using the keywords: CAFs Secretome. The last study was accessed on 29 November 2022. Pharmacological targeting of stromal CAFs can be of clinical advantage in cancers with rich stroma with poor treatment response. Cancer-associated fibroblasts are often associated with drug resistance. Interestingly, CAFs’ effect on drug resistance is context dependent. Understanding the relationship between components of the CAF secretome and the signaling needs of different tumors may help in discovering effective combination treatment strategies. The tumor cell secretome of cancer cells is known to promote chemoresistance, tumor recurrence, and overall poor outcome. In this review, we have summarized studies investigating the CAF secretome and its relevance to cancer. Investigating stromal CAFs as potential therapeutics will help control a pro-tumorous secretome, thereby preventing CAF-mediated cancer progression. However, therapeutic targeting of the CAF secretome remains a challenge and the field and needs to be investigated in detail. The subsections summarize the data available regarding the secretome derived from cancer-specific CAFs and how they impact cancer development, progression, and treatment outcome.Worldwide, colorectal carcinoma ranks third among most diagnosed malignancies and stands second for being the leading cause of death. It is a heterogeneous disease involving different pathways in its development . Colon cFindings from CRC-CAF secretome analysis revealed a hybrid epithelial–mesenchymal transition (EMT) state mediated via hepatocyte growth factor (HGF) signaling. Fibroblasts derived from tumor region in CRC were found to express significantly elevated levels of CAF marker α-SMA . FurtherWorldwide, breast cancer represents the most commonly occurring cancer in women, with a 70–80% cure rate in early-stage, non-metastatic patients. It is a heterogeneous disease . StromalCAF-rich tumor stroma is associated with therapy resistance and inferior prognosis in breast cancer. Hence, it is of great interest to identify stroma-specific novel biomarkers. Experiments have been conducted on conditioned media mimicking tumor stroma interactions for this purpose. Constituents of CAF-conditioned medium were investigated to identify factors that may promote drug resistance in breast cancer cells. Secretome analysis of CAFs-CM exposed to HER2-positive metastatic breast cancer was performed. It was observed that factors from CAF-CM displayed the ability to promote resistance to first-line therapy, trastuzumab with pertuzumab and docetaxel. It was found that CAFs promoted a resistant phenotype in cancer cells via inducing EMT. It was observed that expression of mesenchymal markers, fibronectin and snail, were upregulated in breast cancer cells exposed to CAF-conditioned media . CompariPenile cancer comes under a rare type of genitourinary malignancy which is associated with poor disease outcome and limited therapeutic options. It is of high importance to understand the molecular interactions within the complex TME of penile tumors. Molecular profiling and characterization of deregulated genes and pathways will help in understanding the tumor/tumor microenvironment landscape and will develop novel treatment strategies . SecretoHead and neck squamous cell carcinomas are an aggressive and heterogeneous type of tumor affecting millions of patients worldwide. Despite significant advances in therapeutics over the last several years, the 5-year survival rate has not improved beyond 50%. The involvement of different cells and cell–cell interactions in the complex TME has been implicated in tumor progression and therapy response . SecretoCAFs play critical role in the TME of HCC. CAF-derived factors such as cardiotrophin-like cytokine factor 1 (CLCF1) increased expression of TGF-β and chemokine ligand 6 (CXCL6) secretion from tumor cells. The increased expression further stimulated stemness related features in the tumor cell in an autocrine fashion and increased infiltration of tumor-associated neutrophils (TANs) in a paracrine manner. The CLCF1-CXCL6/TGF-β axis plays a significant role in this cytokine-mediated cellular crosstalk that regulates CAFs, HCC progression, and patient prognosis . InvestiColonization of Helicobacter pylori (Hp) initiates the chain of pathologic events in the gastric mucosa, including local inflammation, followed by gastric ulceration, and development of adenocarcinoma. Infection may also directly and indirectly affect the secretome of the tumor and CAFs. In vitro studies have been carried out to advance understanding regarding the interactions between gastric cells and Hp. Exposing gastric fibroblasts to Hp infection (cagA+vacA+) and studying their impact on normal epithelial cells revealed that conditioned media from Hp-activated gastric fibroblasts instigated EMT-like phenotypic transition and transmigration potential of normal epithelial cells. Gastric fibroblasts are one of the key targets of Hp infection and they communicate via paracrine interactions with epithelial cells and Hp. Hp-activated gastric fibroblasts differentiate toward a CAF-like phenotype, encouraging the EMT-related phenotypic changes in normal gastric epithelial cells . FurtherPDAC counts among one of the most stroma–rich cancers ,61. A seCAFs are known to play a very significant role in OSCC tumorigenesis. OSCC is one of the commonest types of oral cavity tumors . SecretoLike in many other cancers, in OTSCC, a bidirectional crosstalk between CAFs and cancer cells helps in fostering a niche facilitated by secreted factors and various extracellular vesicles. To fully understand this process, patient tumor resected CAFs, isolated and characterized with matched adjacent normal fibroblasts, were used for proteomics analysis. Protein microfibril associated protein 5 (MFAP5) of extracellular microfibrils was found to be enriched in CAF secretomes. Clinical data revealed an association between MFAP5 expression and patient survival, pointing to a role of MFAP5 protein in OTSCC progression .CAFs generated using conditioned media from primary and metastatic melanoma cells displayed elevated motility. CAFs’ secretome analysis revealed enhanced secretion of lactate, upregulated expression of MMPs mediators, several cytokines, including IL6, IL8, CXCL1, CCL2, ICAM1, and proteins associated with angiogenesis, such as VEGFA and GM-CSF. Highly aggressive melanoma cells displayed more prominent effects on the CAFs phenotype in terms of enhanced motility, expressed as an elevated migration and invasion ratio, as well as higher area of digestion in MMP2 and MMP14 proteolytic activity compared to less aggressive melanoma cells . EVs are known for their role as information mediators between tumor and stromal cells. In ovarian cancer, it was observed that EVs released from human ovarian cancer cells were able to activate normal fibroblasts. EVs derived from more aggressive and less aggressive ovarian cancer cells exposed to fibroblasts led to NF activation. The secretome of activated fibroblasts was able to modulate features including proliferation, motility, and invasion of tumor, as well as fibroblasts, and endothelial cells. The findings support the idea that cancer cells can modify fibroblast behavior by release of EVs . CAFs are known to display great heterogeneity in lung cancer and their functional classification is related to clinical response in patients with targeted therapies . CAFs arThe secretome of CAFs represents the assortment of proteins secreted or shed by the cell. The functional and clinical relevance of the secretome of cell populations, including the cancer stem cell , adipocyThe tumor microenvironment is highly complex with heterotypic interactions, hypoxia, inflammation, and redox imbalance. The tumor secretome adds to this complexity . The comCrosstalk between CAFs and hypoxia is a key determinant in the complex immunosuppressive TME . HypoxiaTechnological advancements made in recent years in the field of proteomic analysis have greatly facilitated investigating the secretome in cancers. Broadly, two different approaches are used for secretome analysis. These approaches can be either proteomics-based or can rely on genome-based computational prediction. Proteomic approaches are the core strength of secretome analysis and help biomarker discovery in cancers. Largely, for secretome studies, three different methods of proteomic technologies are used: gel-based, MS-based methods , or SELDI-TOF-MS (surface-enhanced laser desorption/ionization time-of-flight mass spectrometry)-based . All theA comparative proteomic analysis is especially helpful in investigating differential expression of secreted protein from normal fibroblasts and CAFs. Performing 2-D PAGE  and MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry allows for identification of differentially expressed spots. These spots (proteins) play essential roles in tumor development and progression. One such protein identified in the nasopharyngeal carcinoma CAF supernatant was galectin-1 . Using uAn advanced multi-omics platform to identify the biomarkers capable of predicting clinicopathological relevance and improving individualized prognosis based on the secretomic profile is a step in the right direction. Secreto-transcriptomics is a multi-omics technique to find breast cancer subtypes-specific candidate markers of prognostic significance in their oncogenically secreted states. This technique uses label-free quantitative technology to identify proteins showing BC-subtype specific biomarkers. SeCEP, a subtype specific secretion pattern, pinpointed key genes including CDH1, DDB1, CD44, HSPA5, and HSP90B1 involved in an oncogenically active secretome. The SeCEP gene data allowed identification of novel liquid biopsy biomarkers for prediction of advanced individualized prognosis . SILAC-bThe field of secretome analysis has gained traction in recent years because of various advancements in proteomic technology. Establishing a clear and reliable method for sample collection and preparation for secretome analysis is still challenging, however, the major limitation with CAFs secretome analysis is the heterogeneity in the CAF population itself. Multiple factors impacting the diversity, such as the origin of CAFs, activation state, and response to stress, may encourage functional heterogeneity . FurtherThe sample preparation for secretome analysis is especially challenging. To investigate the cancer secretome, biological fluids in the tumor proximity or the interstitial fluid in the tumor region should be studied to estimate the proteins present. Using conditioned media samples derived from cancer cell lines is more commonly used for discoveries related to the cancer secretome. However, it has been found that conditioned media derived from in vitro cultured cells is significantly less complex compared to their biological fluid counterparts . FurtherInvestigating a CAFs-derived secretome offers advantages to establish a CAF-specific profile. Further, studying the CAFs secretome will help in identification of clinically relevant biomarkers for CAF-dense cancer types. Investigating a conditioned medium from cancer cells and CAFs allows the identification of the assortment of secreted proteins promoting cancer proliferation and metastasis. Some of these proteins may also be useful in designing serum-based tests for early and non-invasive detection of cancer. Secretome profiling and analysis will speed up the process of diagnosis, prognosis, and monitoring of cancer . Studies based on secretome profiling and analysis will support an efficient and personalized cancer care by adding parameters to address tumor recurrence, chemoresistance, and overall disease prognosis. However, certain limitations regarding the standardization process for the formulation of cell-conditioned media, characterization of biologically active molecules in the conditioned media, and the mode of action need to be overcome. Taken together, a deeper understanding of underlying factors that may introduce variability in the composition of the CAF-derived secretome needs to be investigated further. Through this review, we have focused on the broad contribution of the CAFs secretome towards inducing a pro-tumorigenic, immunomodulatory environment capable of restoring aberrant cancer cell growth and progression. Direct application of the CAFs secretome in the cancer therapeutic arena holds great promise but still has a long way to go. Nonetheless, having an advanced knowledge of the CAFs secretome will help in better management of stromal CAFs-rich cancers. Using this information for designing a combination therapy is of high clinical interest and relevance in the future.


Upper gastrointestinal (GI) malignancy is a leading cause of cancer-related morbidity and mortality. Upper endoscopy has an established role in diagnosing and staging upper GI cancers, screening for pre-malignant lesions, and providing palliation in cases of advanced malignancy. New advances in endoscopic techniques and technology have improved diagnostic accuracy and increased the therapeutic potential of upper endoscopy. We aim to describe the different types of endoscopic technology used in cancer diagnosis, summarize the current guidelines for endoscopic diagnosis and treatment of malignant and pre-malignant lesions, and explore new potential roles for endoscopy in cancer therapy. Since the inception of the flexible endoscope in the 1950s, gastrointestinal (GI) endoscopy has played an ever-increasing role in the diagnosis of upper GI tract cancers and screening of pre-cancerous lesions ,3. Conventional video endoscopy is the gold standard for diagnosing a wide range of upper GI malignancies. Chromoendoscopy, the intra-procedural application of stains and dyes to highlight abnormal mucosa, has traditionally been used to supplement video endoscopy and improve diagnostic yield . AdvanceIn addition to direct visualization of luminal masses, evaluation of suspicious lesions in the gastrointestinal wall and extraluminal structures such as the pancreas can be performed via endoscopic ultrasound (EUS), and direct access to the biliary or pancreatic ducts for biopsy and palliative stent placement can be performed using endoscopic retrograde cholangiopancreatography (ERCP) ,10,11,12This article aims to explore the diagnostic and therapeutic function of endoscopy in several subtypes of upper GI cancers, and provides a brief review of new developments.Barrett’s esophagus (BE), defined as >1 cm of columnar epithelium with intestinal metaplasia in the distal esophagus, is a known risk factor for development of esophageal adenocarcinoma (EAC) . ScreeniPatients with columnar epithelium seen on endoscopy require histological confirmation of intestinal metaplasia in order to diagnose BE. Additional lesions that appear nodular, ulcerated, or flat with irregular mucosal contours should be resected via endoscopic mucosa resection (EMR) or endoscopic submucosal dissection (ESD) for histologic evaluation ,17. ThesBE surveillance is performed with HD endoscopy, electronic chromoendoscopy (most commonly via narrow-band imaging [NBI]), and biopsy, and screening interval depends on degree of dysplasia see  16,17].,17.16,17Esophageal epidermoid metaplasia (EEM) is a new entity that is histologically analogous to leukoplakia in the mouth . It is gGastric intestinal metaplasia (GIM) is a transitional stage in the linear progression from gastritis to gastric carcinoma ,27. EndoScreening guidelines for pre-cancerous gastric lesions like GIM differ by country depending on the relative incidence of gastric cancer. In South Korea, the country with the highest age-standardized incidence of gastric cancer in the world, the National Cancer Screening Program (NCSP) offers screening with either endoscopy or upper-GI series every two years for all individuals between ages 40–75 . StudiesH. pylori screening with subsequent treatment if they test positive . Th. Th39]. Treatment for MCNs and IPMNs with high-grade dysplasia or carcinoma is surgical resection; however, the risks and benefits of surgery should be evaluated on a case-by-case basis, since older patients with other comorbidities died more frequently of causes unrelated to their pancreatic neoplasm . MCNs wiDuodenal adenomas can be categorized by location as ampullary or non-ampullary . Non-ampHowever, individuals with familial adenomatous polyposis (FAP) have approximately a 5% lifetime risk of developing duodenal carcinoma and require regular surveillance ,49. FAP Screening and surveillance are performed endoscopically; the side-viewing endoscope and ERCP can be useful for visualizing the ampulla and evaluating for biliary and pancreatic duct involvement ,52. AddiEGD is indicated for patients with risk factors and symptoms concerning for upper GI malignancy, such as persistent dyspepsia, esophageal dysphagia or vomiting in patients older than 50 years, anorexia, weight loss, early satiety, and unexplained iron deficiency anemia . EndoscoTumors are most common staged according to the TNM  system. This evaluation begins with CT imaging, since the presence of metastatic disease precludes curative tumor resection ,62. In pEUS can inform N-staging by visualization locoregional lymph nodes or by lymph node biopsy via EUS-FNA. Four characteristics of lymph nodes seen on EUS—size > 10 mm, round shape, sharp borders, and absence of central intranodal vessels—are traditionally associated with a higher likelihood of malignancy, although its accuracy is poor  ,63. StudPancreatic cancer is most commonly diagnosed via initial CT imaging; equivocal imaging results can often be clarified with EUS-FNA, which has been shown to have sensitivity and specificity greater than 90% . StudiesCholangiocarcinoma is traditionally diagnosed with cross-sectional imaging (MRCP is preferred), although distal extrahepatic neoplasms may be diagnosed via EUS-FNA or endoscopic retrograde cholangiography (ERC). Fluoroscopy-guided shaped endobiliary biopsy (FSEB) is a newer technique that involves manually re-shaping the endoscopy forceps to permit easier access to a biliary stricture, and has been shown to have high sensitivity for diagnosing both proximal and distal biliary neoplasms . IntraduGallbladder cancer is a rare entity that is diagnosed incidentally after cholecystectomy in approximately 50% of cases . IndividStaging of pancreaticobiliary cancers generally begins with cross-sectional imaging, similar to the case with intraluminal cancers ,78,81. SAs mentioned above, stage T1a esophageal carcinoma in the absence of metastasis can be treated endoscopically, with the goal of achieving R0 resection  ,82. StudGastric adenocarcinoma makes up the overwhelming majority of gastric cancer cases, and most of the endoscopic treatment guidelines come from countries with a high prevalence, such as Japan and Korea. In general, well-differentiated tumors that are limited to the mucosa without ulceration, ≤2 cm without evidence of nodal or distant metastasis should be treated with EMR ,85. WellPatients found to have lympho-vascular invasion, positive vertical margins, or greater than 500μm tumor extension into the submucosa are at high rate for nodal metastasis and should be treated with gastrectomy ,85. In cIn addition to adenocarcinoma, gastric neuroendocrine tumor (G-NET) is another type of gastric cancer that is being detected at increasing frequency ,88,89. GSurgical resection was traditionally preferred over endoscopic therapy as the first line treatment for patients with resectable gastrointestinal stromal tumors (GIST) of the stomach, since these malignancies originate in the deeper muscularis propria layer . HoweverPancreatic cysts are normally treated with surgical resection, and adenocarcinoma (PDAC) or neuroendocrine tumors (PNET) are treated with surgery (if resectable) or systemic chemoradiation. In recent years, endoscopic ablative procedures have been used with increasing frequency ,97. A smSimilar to pancreatic cancer, resectable ampullary adenocarcinoma is typically treated surgically, and ampullectomy or pancreaticoduodenectomy currently remain the standard of care . HoweverEndoscopic radiofrequency ablation (RFA) is a relatively new technique that promising outcomes in patients with malignant biliary obstruction who are not candidates for surgery . When usPatients with advanced upper gastrointestinal cancer are usually not candidates for curative therapy, and management of their condition is tailored toward palliative treatment . DysphagStenting is the standard of care for treating obstruction in the esophagus, gastric outlet, and pancreatic and biliary ducts. This procedure has a high rate of technical success with a short post-procedure length of hospital stay, and improves nutritional status, obstructive symptoms, and quality of life ,106,107.The most common types of stents are self-expandable metal stents (SEMS) and plastic stents. SEMS are preferred in most cases due to a lower rate of complications ,110, altIn cases of gastric outlet obstruction (GOO), surgical gastrojejunostomy can be performed in lieu of stenting. Surgery is associated with longer hospitalization and time to resume oral intake, but has fewer long-term complications requiring re-intervention . In receAlthough patients with advanced upper gastrointestinal cancer are often malnourished, current guidelines recommend against routine nutritional support for cachectic patients with untreatable or advanced cancer due to the risk of procedure-related complications and lack of mortality benefit . HoweverThe most common techniques for enteral feeding tube placement are percutaneous endoscopic gastrostomy (PEG) or percutaneous radiographic gastrostomy (PRG); surgical feeding tube placement is less preferred because it is more invasive . HoweverDetermining which patients would benefit from enteral feeding and the modality of tube placement should be an individualized decision that takes into account the patient’s nutritional status and prognosis, as well as technical procedural factors; therefore, the decision of feeding placement is best made after a multidisciplinary discussion ,114,118.EUS-guided celiac plexus block (CPB) and neurolysis (CPN) are minimally invasive palliative modalities that can be used to manage malignancy-related pain, usually from pancreatic cancer, that is refractory to medical management . CPB proThe aforementioned techniques and technologic advances have transformed the care of patients with upper GI cancers. Advances in endoscopic visualization have led to earlier identification of pre-malignant lesions ,131. AddDespite these advances, several limitations of upper endoscopy remain. The role of endoscopic therapeutic techniques is currently restricted to small, early-stage tumors and palliation. Tumor location may also precludes endoscopic treatment due to the risk of post-procedure structuring following EMR or ESD. Novel techniques, such as RFA in pancreatic neuroendocrine tumors or EFTR for resection of gastrointestinal stromal tumors, have shown promise in small studies but still need to be validated in larger clinical trials. Additionally, surveillance guidelines for gastric intestinal metaplasia and pancreatic cysts are limited by low quality of evidence. Further studies in these areas are needed to optimize cancer screening and preventative care for these patients.There is a growing body of literature on deep learning in medicine in the past decade . In regaAdvances in endoscopy have led to an ever-increasing role in cancer treatment. One prominent example of this is in the area of endoscopically guided injection of anti-tumor therapies. Endoscopic administration of 5-FU in late-stage esophageal cancer was reported in the 1990s, although clinical benefit was limited . Since tAdvancements in endoscopy have provided highly accurate, non-invasive methods for detecting upper GI malignancy and pre-malignant lesions. Recent developments in endoscopic technique have made endoscopic treatments the preferred technique for resection of small focal lesions and palliative management of non-resectable malignancies, and ongoing studies offer the promise of an even greater role for the endoscopic oncologist.