Cellular Turnover Research Articles

Tissue engineered bovine saphenous vein extracellular matrix scaffolds produced via antigen removal achieve high in vivo patency rates

Diseases of small diameter blood vessels encompass the largest portion of cardiovascular diseases, with over 4.2 million people undergoing autologous vascular grafting every year. However, approximately one third of patients are ineligible for autologous vascular grafting due to lack of suitable donor vasculature. Acellular extracellular matrix (ECM) scaffolds derived from xenogeneic vascular tissue have potential to serve as ideal biomaterials for production of off-the-shelf vascular grafts capable of eliminating the need for autologous vessel harvest. A modified antigen removal (AR) tissue process, employing aminosulfabetaine-16 (ASB-16) was used to create off-the-shelf small diameter (< 3 mm) vascular graft from bovine saphenous vein ECM scaffolds with significantly reduced antigenic content, while retaining native vascular ECM protein structure and function. Elimination of native tissue antigen content conferred graft-specific adaptive immune avoidance, while retention of native ECM protein macromolecular structure resulted in pro-regenerative cellular infiltration, ECM turnover and innate immune self-recognition in a rabbit subpannicular model. Finally, retention of the delicate vascular basement membrane protein integrity conferred endothelial cell repopulation and 100% patency rate in a rabbit jugular interposition model, comparable only to Autograft implants. Alternatively, the lack of these important basement membrane proteins in otherwise identical scaffolds yielded a patency rate of only 20%. We conclude that acellular antigen removed bovine saphenous vein ECM scaffolds have potential to serve as ideal off-the-shelf small diameter vascular scaffolds with high in vivo patency rates due to their low antigen content, retained native tissue basement membrane integrity and preserved native ECM structure, composition and functional properties. Statement of significanceThe use of autologous vessels for the treatment of small diameter vascular diseases is common practice. However, the use of autologous tissue poses significant complications due to tissue harvest and limited availability. Developing an alternative vessel for use for the treatment of small diameter vessel diseases can potentially increase the success rate of autologous vascular grafting by eliminating complications related to the use of autologous vessel and increased availability. This manuscript demonstrates the potential of non-antigenic extracellular matrix (ECM) scaffolds derived from xenogeneic vascular tissue as off-the-shelf vascular grafts for the treatment of small diameter vascular diseases.

Proteoglycan synthesis in conserved oligomeric Golgi subunit deficient HEK293T cells is affected differently, depending on the lacking subunit.

The Conserved Oligomeric Golgi (COG) complex is an eight subunit protein complex associated with Golgi membranes. Genetic defects affecting individual COG subunits cause congenital disorders of glycosylation (CDGs), due to mislocalization of Golgi proteins involved in glycosylation mechanisms. While the resulting defects in N-and O-glycosylation have been extensively studied, no corresponding study of proteoglycan (PG) synthesis has been undertaken. We here show that glycosaminoglycan (GAG) modification of PGs is significantly reduced, regardless which COG subunit that is missing in HEK293T cells. Least reduction was observed for cells lacking COG1 and COG8 subunits, that bridge the A and B lobes of the complex. Lack of these subunits did not reduce GAG chain lengths of secreted PGs, which was reduced in cells lacking any other subunit (COG2-7). COG3 knock out (KO) cells had particularly reduced ability to polymerize GAG chains. For cell-associated GAGs, the mutant cell lines, except COG4 and COG7 KO, displayed longer GAG chains than wild-type cells, indicating that COG subunits play a role in cellular turnover of PGs. In light of the important roles PGs play in animal development, the effects KO of individual COG subunits have on GAG synthesis could explain the variable severity of COG associated CDGs.

Global transcriptome profiling reveals antagonizing response of head kidney of juvenile common carp exposed to glyphosate

Glyphosate (GLY) frequently detected in various water bodies has imposed a serious risk on fish. Head kidney of fish is an important defense organ, playing a vital part in antagonizing exogenous hazardous matter. The objective of this study was to characterize toxic mechanisms of GLY in head kidney of common carp based on transcriptome profiling. After 45-days exposure of GLY at environmentally relevant concentrations, juvenile common carp were used as experimental subjects to analyze how the head kidney responded to GLY. The transcriptome profiling identified 1381 different expressed genes (DEGs) between the control and exposure groups (5 and 50 mg/L). Functional analysis of DEGs substantiated over-representative pathways mainly involving cellular stress responses, cell proliferation and turnover, apoptosis, lipid metabolism, and innate immune processes in both treated groups compared with the control group. Predicted network of gene regulation indicated that GLY-induced tp53 played a vital role in linking a battery of signals. Furthermore, the expression of 10 candidate genes by qRT-PCR aligned with transcriptional profiling. In addition, western blotting analysis confirmed that GLY-induced apoptosis and cellular proliferation were closely involved in activating MAKP signaling pathway and lipid metabolism pathway in both treated groups. Collectively, these data demonstrate that head kidney of juvenile common carp mainly leverages upregulation of genes related to cell proliferation and turnover, apoptosis, and lipid metabolism to combat sub-chronic exposure of GLY. This study casts new understanding into the risk of GLY in aquatic animals.

Snow buntings preparing for migration increase muscle fiber size and myonuclear domain in parallel with a major gain in fat mass

In long‐distance migrants, preparation for migration is typically associated with increases in fat and body mass, and with an enlargement of pectoralis muscle mass that likely improves flight performance. Although changes in muscle mass or size have been well described in migratory birds, potential changes in muscle ultrastructure during this transition still deserves scrutiny. Using outdoor captive snow buntings (Plectrophenax nivalis n = 15) measured during their transition into a spring migratory phenotype as a model system, we studied changes in pectoralis muscle ultrastructure and predicted that muscle fiber diameter could increase in parallel with the gain in body mass. We also expected that larger fibers could either recruit satellite cells to support cellular maintenance and protein turnover, increase myonuclear domain (cytoplasm per nuclei) with a potential increase in protein turnover load per myonucleus, or existing myonuclei could undergo endoreduplication. Buntings increased body mass by 46% within a month, largely due to a &gt; 6‐fold increase in body fat. However, this increase in body mass was also associated with a 36% increase in muscle fiber diameter. Both pectoralis muscle mass (r2 = 0.57–0.77) and fiber diameter (r2 = 0.32) correlated with total body mass, without any change in the number of nuclei per fiber. Consequently, variation in myonuclear domain (i.e. the amount of cytoplasm per nucleus), was also positively associated with body mass (r2 = 0.51). Therefore, buntings preparing for migration may experience an increase in muscle contraction force due to larger muscle fibers, but this is also coupled with increases in myonuclear domain, which may force these cells to increase protein production to safeguard satellite cells.

Oxidative stress sensing and response in neural stem cell fate

Neural stem/progenitor cells (NSPCs) contribute to the physiological cellular turnover of the adult brain and make up its regenerative potential. It is thus essential to understand how different factors influence their proliferation and differentiation to gain better insight into potential therapeutic targets in neurodegenerative diseases and traumatic brain injuries. Recent evidences indicate the roles of redox stress sensing and coping mechanisms in mediating the balance between NSPC self-renewal and differentiation. Such mechanisms involve direct cysteine modification, signaling and metabolic reprogramming, epigenetic alterations and transcription changes leading to adaptive responses like autophagy. Here, we discuss emerging findings on the involvement of redox sensors and effectors and their mechanisms in influencing changes in cellular redox potential and NSPC fate.

Self-renewal of double-negative 3 early thymocytes enables thymus autonomy but compromises the β-selection checkpoint

T lymphocyte differentiation in the steady state is characterized by high cellular turnover whereby thymocytes do not self-renew. However, if deprived of competent progenitors, the thymus can temporarily maintain thymopoiesis autonomously. This bears a heavy cost, because prolongation of thymus autonomy causes leukemia. Here, we show that, at an early stage, thymus autonomy relies on double-negative 3 early (DN3e) thymocytes that acquire stem-cell-like properties. Following competent progenitor deprivation, DN3e thymocytes become long lived, are required for thymus autonomy, differentiate invivo, and include DNA-label-retaining cells. At the single-cell level, the transcriptional programs of thymopoiesis in autonomy and the steady state are similar. However, a new cell population emerges in autonomy that expresses an aberrant Notch target gene signature and bypasses the β-selection checkpoint. In summary, DN3e thymocytes have the potential to self-renew and differentiate invivo if cell competition is impaired, but this generates atypical cells, probably the precursors of leukemia.

Epigenetic Regulation of Cell-Fate Changes That Determine Adult Liver Regeneration After Injury.

The adult liver has excellent regenerative potential following injury. In contrast to other organs of the body that have high cellular turnover during homeostasis (e.g., intestine, stomach, and skin), the adult liver is a slowly self-renewing organ and does not contain a defined stem-cell compartment that maintains homeostasis. However, tissue damage induces significant proliferation across the liver and can trigger cell-fate changes, such as trans-differentiation and de-differentiation into liver progenitors, which contribute to efficient tissue regeneration and restoration of liver functions. Epigenetic mechanisms have been shown to regulate cell-fate decisions in both embryonic and adult tissues in response to environmental cues. Underlying their relevance in liver biology, expression levels and epigenetic activity of chromatin modifiers are often altered in chronic liver disease and liver cancer. In this review, I examine the role of several chromatin modifiers in the regulation of cell-fate changes that determine efficient adult liver epithelial regeneration in response to tissue injury in mouse models. Specifically, I focus on epigenetic mechanisms such as chromatin remodelling, DNA methylation and hydroxymethylation, and histone methylation and deacetylation. Finally, I address how altered epigenetic mechanisms and the interplay between epigenetics and metabolism may contribute to the initiation and progression of liver disease and cancer.

Tuning flavin environment to detect and control light-induced conformational switching in Drosophila cryptochrome

Light-induction of an anionic semiquinone (SQ) flavin radical in Drosophila cryptochrome (dCRY) alters the dCRY conformation to promote binding and degradation of the circadian clock protein Timeless (TIM). Specific peptide ligation with sortase A attaches a nitroxide spin-probe to the dCRY C-terminal tail (CTT) while avoiding deleterious side reactions. Pulse dipolar electron-spin resonance spectroscopy from the CTT nitroxide to the SQ shows that flavin photoreduction shifts the CTT ~1 nm and increases its motion, without causing full displacement from the protein. dCRY engineered to form the neutral SQ serves as a dark-state proxy to reveal that the CTT remains docked when the flavin ring is reduced but uncharged. Substitutions of flavin-proximal His378 promote CTT undocking in the dark or diminish undocking in the light, consistent with molecular dynamics simulations and TIM degradation activity. The His378 variants inform on recognition motifs for dCRY cellular turnover and strategies for developing optogenetic tools.

Defence and adaptation mechanisms of the intestinal epithelium upon infection

The intestinal epithelium is a monolayer of polarized columnar cells that act as a border between the host and its environment and are the first line of defence against the luminal microbes. In addition to providing a physical barrier, the epithelium possesses a multitude of active mechanisms to fight invading pathogens and regulate the composition and spatial distribution of commensals. The different epithelial cell types have unique functions in this context, and crosstalk with the immune system further modulates their intricate antimicrobial responses.The epithelium is organized into clonal crypt units with a high cellular turnover that is driven by stem cells located at the base. There is increasing evidence that this anatomical organization, the stem cell turnover, and the lineage determination processes are essential for barrier maintenance. These processes can be modulated by microbes directly or by the immune responses to enteric pathogens, resulting in a rapid and efficient adaptation of the epithelium to environmental perturbations, injuries, and infections. Here we discuss the complex host-microbial interactions that shape the mucosa and how the epithelium maintains and re-establishes homeostasis after infection.

Determining the stages of cellular differentiation using deep ultraviolet resonance Raman spectroscopy

Cellular differentiation is a fundamental process in which one cell type changes into one or more specialized cell types. Cellular differentiation starts at the beginning of embryonic development when a simple zygote begins to transform into a complex multicellular organism composed of various cell and tissue types. This process continues into adulthood when adult stem cells differentiate into more specialized cells for normal growth, regeneration, repair, and cellular turnover. Any abnormalities associated with this fundamental process of cellular differentiation are linked to life-threatening conditions, including degenerative diseases and cancers. Detection of undifferentiated and different stages of differentiated cells can be used for disease diagnosis but is often challenging due to the laborious procedures, expensive tools, and specialized technical skills which are required. Here, a novel approach, called deep ultraviolet resonance Raman spectroscopy, is used to study various stages of cellular differentiation using a well-known myoblast cell line as a model system. These cells proliferate in the growth medium and spontaneously differentiate in differentiation medium into myocytes and later into myotubes. The cellular and molecular characteristics of these cells mimic very well actual muscle tissue in vivo. We have found that undifferentiated myoblast cells and myoblast cells differentiated at three different stages are able to be easily separated using deep ultraviolet resonance Raman spectroscopy in combination with chemometric techniques. Our study has a great potential to study cellular differentiation during normal development as well as to detect abnormal cellular differentiation in human pathological conditions in future studies.

The Distinct Function of p21Waf1/Cip1 With p16Ink4a in Modulating Aging Phenotypes of Werner Syndrome by Affecting Tissue Homeostasis

Human Werner syndrome (WS) is an autosomal recessive progeria disease. A mouse model of WS manifests the disease through telomere dysfunction-induced aging phenotypes, which might result from cell cycle control and cellular senescence. Both p21Waf1/Cip1 (p21, encoded by the Cdkn1a gene) and p16Ink4a (p16, encoded by the Ink4a gene) are cell cycle inhibitors and are involved in regulating two key pathways of cellular senescence. To test the effect of p21 and p16 deficiencies in WS, we crossed WS mice (DKO) with p21–/– or p16–/– mice to construct triple knockout (p21-TKO or p16-TKO) mice. By studying the survival curve, bone density, regenerative tissue (testis), and stem cell capacity (intestine), we surprisingly found that p21-TKO mice displayed accelerated premature aging compared with DKO mice, while p16-TKO mice showed attenuation of the aging phenotypes. The incidence of apoptosis and cellular senescence were upregulated in p21-TKO mice tissue and downregulated in p16-TKO mice. Surprisingly, cellular proliferation in p21-TKO mice tissue was also upregulated, and the p21-TKO mice did not show telomere shortening compared with age-matched DKO mice, although p16-TKO mice displayed obvious enhancement of telomere lengthening. Consistent with these phenotypes, the SIRT1-PGC1 pathway was upregulated in p16-TKO but downregulated in p21-TKO compared with DKO mouse embryo fibroblasts (MEFs). However, the DNA damage response pathway was highly activated in p21-TKO, but rescued in p16-TKO, compared with DKO MEFs. These data suggest that p21 protected the stem cell reservoir by regulating cellular proliferation and turnover at a proper rate and that p21 loss in WS activated fairly severe DNA damage responses (DDR), which might cause an abnormal increase in tissue homeostasis. On the other hand, p16 promoted cellular senescence by inhibiting cellular proliferation, and p16 deficiency released this barrier signal without causing severe DDR.

Targeting the Hippo Pathway in Prostate Cancer: What's New?

Simple SummaryProstate cancer is the most commonly diagnosed cancer in men in the UK, accounting for the deaths of over 11,000 men per year. A major problem in this disease are tumours which no longer respond to available treatments. Understanding how this occurs will reveal new ways to treat these patients. In this review, the latest findings regarding a particular group of cellular factors which make up a signalling network called the Hippo pathway will be described. Accumulating evidence suggests that this network contributes to prostate cancer progression and resistance to current treatments. Identifying how this pathway can be targeted with drugs is a promising area of research to improve the treatment of prostate cancer.Identifying novel therapeutic targets for the treatment of prostate cancer (PC) remains a key area of research. With the emergence of resistance to androgen receptor (AR)-targeting therapies, other signalling pathways which crosstalk with AR signalling are important. Over recent years, evidence has accumulated for targeting the Hippo signalling pathway. Discovered in Drosophila melanogasta, the Hippo pathway plays a role in the regulation of organ size, proliferation, migration and invasion. In response to a variety of stimuli, including cell–cell contact, nutrients and stress, a kinase cascade is activated, which includes STK4/3 and LATS1/2 to inhibit the effector proteins YAP and its paralogue TAZ. Transcription by their partner transcription factors is inhibited by modulation of YAP/TAZ cellular localisation and protein turnover. Trnascriptional enhanced associate domain (TEAD) transcription factors are their classical transcriptional partner but other transcription factors, including the AR, have been shown to be modulated by YAP/TAZ. In PC, this pathway can be dysregulated by a number of mechanisms, making it attractive for therapeutic intervention. This review looks at each component of the pathway with a focus on findings from the last year and discusses what knowledge can be applied to the field of PC.

Deciphering the miRNA transcriptome of breast muscle from the embryonic to post-hatching periods in chickens

BackgroundmiRNAs play critical roles in growth and development. Various studies of chicken muscle development have focused on identifying miRNAs that are important for embryo or adult muscle development. However, little is known about the role of miRNAs in the whole muscle development process from embryonic to post-hatching periods. Here, we present a comprehensive investigation of miRNA transcriptomes at 12-day embryo (E12), E17, and day 1 (D1), D14, D56 and D98 post-hatching stages.ResultsWe identified 337 differentially expressed miRNAs (DE-miRNAs) during muscle development. A Short Time-Series Expression Miner analysis identified two significantly different expression profiles. Profile 4 with downregulated pattern contained 106 DE-miRNAs, while profile 21 with upregulated pattern contained 44 DE-miRNAs. The DE-miRNAs with the upregulated pattern mainly played regulatory roles in cellular turnover, such as pyrimidine metabolism, DNA replication, and cell cycle, whereas DE-miRNAs with the downregulated pattern directly or indirectly contributed to protein turnover metabolism such as glycolysis/gluconeogenesis, pyruvate metabolism and biosynthesis of amino acids.ConclusionsThe main functional miRNAs during chicken muscle development differ between embryonic and post-hatching stages. miRNAs with an upregulated pattern were mainly involved in cellular turnover, while miRNAs with a downregulated pattern mainly played a regulatory role in protein turnover metabolism. These findings enrich information about the regulatory mechanisms involved in muscle development at the miRNA expression level, and provide several candidates for future studies concerning miRNA-target function in regulation of chicken muscle development.

Higher serum uric acid is associated with higher risks of thrombosis and death in patients with primary myelofibrosis

Serum uric acid (SUA) can promote inflammation and is associated with increased cardiovascular morbidity. Primary (PMF) and secondary myelofibrosis (SMF) are myeloproliferative neoplasms characterized by high cellular turnover and substantial risk of thrombosis and death. We have retrospectively investigated SUA in 173patients with myelofibrosis (125 PMF; 48SMF) and 30controls. The PMF patients had significantly higher SUA in comparison to SMF and controls. In both PMF and SMF higher SUA was significantly associated with arterial hypertension and decreased renal function. Among PMF patients, higher SUA was significantly associated with older age, larger spleen, higher white blood cell counts, higher lactate dehydrogenase, lower immunoglobulin G levels, allopurinol use and non-smoking. Among SMF patients, higher SUA was associated with male sex (P < 0.05 for all analyses). In PMF higher SUA was univariately associated with inferior survival (> 427 μmol/L hazard ratio (HR) = 2.22; P = 0.006) and shorter time to thrombosis (> 444 μmol/L HR = 5.05; P = 0.006), which could be shown separately for arterial (> 380 μmol/L; HR = 4.9; P = 0.013) and venous thromboses (> 530 μmol/L; HR = 17.9; P < 0.001). In multivariate analyses, SUA remained significantly associated with inferior survival independent of the Dynamic International Prognostic Staging System and with shorter time to thrombosis independent of age in PMF patients; however, the prognostic significance of SUA was diminished after including serum creatinine in the models. SUA was not prognostic in SMF patients. The PMF patients present with higher SUA levels, which are associated with features of more advanced disease and higher risks of arterial and venous thrombosis and death.

Relationship between CD4 T cell turnover, cellular differentiation and HIV persistence during ART.

The precise role of CD4 T cell turnover in maintaining HIV persistence during antiretroviral therapy (ART) has not yet been well characterized. In resting CD4 T cell subpopulations from 24 HIV-infected ART-suppressed and 6 HIV-uninfected individuals, we directly measured cellular turnover by heavy water labeling, HIV reservoir size by integrated HIV-DNA (intDNA) and cell-associated HIV-RNA (caRNA), and HIV reservoir clonality by proviral integration site sequencing. Compared to HIV-negatives, ART-suppressed individuals had similar fractional replacement rates in all subpopulations, but lower absolute proliferation rates of all subpopulations other than effector memory (TEM) cells, and lower plasma IL-7 levels (p = 0.0004). Median CD4 T cell half-lives decreased with cell differentiation from naïve to TEM cells (3 years to 3 months, p<0.001). TEM had the fastest replacement rates, were most highly enriched for intDNA and caRNA, and contained the most clonal proviral expansion. Clonal proviruses detected in less mature subpopulations were more expanded in TEM, suggesting that they were maintained through cell differentiation. Earlier ART initiation was associated with lower levels of intDNA, caRNA and fractional replacement rates. In conclusion, circulating integrated HIV proviruses appear to be maintained both by slow turnover of immature CD4 subpopulations, and by clonal expansion as well as cell differentiation into effector cells with faster replacement rates.

The distribution of cellular turnover in the human body.

We integrated ubiquity, mass and lifespan of all major cell types to achieve a comprehensive quantitative description of cellular turnover. We found a total cellular mass turnover of 80 ± 20 grams per day, dominated by blood cells and gut epithelial cells. In terms of cell numbers, close to 90% of the (0.33 ± 0.02) × 1012 cells per day turnover was blood cells.

Tissue Engineered Bovine Saphenous Vein Extracellular Matrix Scaffolds Produced Via Antigen Removal Achieve High &lt;i&gt;In Vivo&lt;/i&gt; Patency Rates

Diseases of small diameter blood vessels encompass the largest portion of cardiovascular diseases, with over 4.2 million people undergoing autologous vascular grafting every year. However, approximately one third of patients are ineligible for autologous vascular grafting due to lack of suitable donor vasculature. Acellular extracellular matrix (ECM) scaffolds derived from xenogeneic vascular tissue have potential to serve as ideal biomaterials for production of an off-the-shelf vascular graft capable of eliminating the need for autologous vessel harvest. Using the antigen removal tissue processing method an off-the-shelf small diameter (< 3 mm) vascular graft from bovine saphenous vein was produced with significantly reduced antigenic content, while retaining the structure and function of its native vascular ECM proteins. Elimination of native tissue antigen content conferred graft-specific adaptive immune avoidance. Retention of native ECM protein macromolecular structure, as assessed by collagen polarization, resulted in pro-regenerative cellular infiltration, ECM turnover and innate immune self-recognition. Finally, retention of the delicate vascular basement membrane protein integrity conferred endothelial cell repopulation and 100% patency rate in a rabbit jugular interposition model, comparable only to Autograft implants. Alternatively, the lack of these important basement membrane proteins in otherwise identical scaffolds yielded a patency rate of only 20%. We conclude that acellular antigen removed bovine saphenous vein ECM scaffolds have potential to serve as ideal off-the-shelf small diameter vascular scaffolds with high in vivo patency rates due to their low antigen content, retained native tissue basement membrane integrity and preserved native ECM structure, composition and functional properties.

Ion Channels, Transporters, and Sensors Interact with the Acidic Tumor Microenvironment to Modify Cancer Progression.

Solid tumors, including breast carcinomas, are heterogeneous but typically characterized by elevated cellular turnover and metabolism, diffusion limitations based on the complex tumor architecture, and abnormal intra- and extracellular ion compositions particularly as regards acid-base equivalents. Carcinogenesis-related alterations in expression and function of ion channels and transporters, cellular energy levels, and organellar H+ sequestration further modify the acid-base composition within tumors and influence cancer cell functions, including cell proliferation, migration, and survival. Cancer cells defend their cytosolic pH and HCO3- concentrations better than normal cells when challenged with the marked deviations in extracellular H+, HCO3-, and lactate concentrations typical of the tumor microenvironment. Ionic gradients determine the driving forces for ion transporters and channels and influence the membrane potential. Cancer and stromal cells also sense abnormal ion concentrations via intra- and extracellular receptors that modify cancer progression and prognosis. With emphasis on breast cancer, the current review first addresses the altered ion composition and the changes in expression and functional activity of ion channels and transporters in solid cancer tissue. It then discusses how ion channels, transporters, and cellular sensors under influence of the acidic tumor microenvironment shape cancer development and progression and affect the potential of cancer therapies.

Genotype-Specific Differences in Circulating Tumor DNA Levels in Advanced NSCLC

IntroductionPlasma-based circulating tumor DNA (ctDNA) is an established biomarker for molecular profiling with emerging applications in disease monitoring in multiple tumor types, including, NSCLC. However, determinants of ctDNA shedding and correlation with tumor burden are incompletely understood, particularly in advanced-stage disease. MethodsWe retrospectively analyzed ctDNA-based and tissue-based genomic data and imaging from 144 patients with NSCLC. Tumor burden was quantified with computed tomography (CT) and brain magnetic resonance imaging for the overall cohort and 18F-fludeoxyglucose positron emission tomography-CT in a subset of patients. ResultsThere was a moderate but statistically significant correlation between ctDNA variant allele frequency and multiple imaging measures of tumor burden such as CT volume (rho = 0.34, p ≤ 0.0001) and metabolic tumor volume (rho = 0.36, p = 0.003). This correlation was strongest in KRAS-mutant tumors (rho = 0.56, p ≤ 0.001), followed by TP53 mutants (rho = 0.43, p ≤ 0.0001), and weakest in EGFR-mutated (EGFR+) tumors (rho = 0.24, p = 0.077). EGFR+ tumors with EGFR copy number gain had significantly higher variant allele frequency than EGFR+ without copy number gain (p ≤ 0.00001). In multivariable analysis, TP53 and EGFR mutations, visceral metastasis, and tumor burden were independent predictors of increased ctDNA shedding. ConclusionsLevels of detectable ctDNA were affected not only by tumor burden but also by tumor genotype. The genotype-specific differences observed may be due to variations in DNA shedding and cellular turnover. These findings have implications for the emerging use of ctDNA in NSCLC disease monitoring and early detection.

Label-free metabolic clustering through unsupervised pixel classification of multiparametric fluorescent images

Autofluorescence microscopy is a promising label-free approach to characterize NADH and FAD metabolites in live cells, with potential applications in clinical practice. Although spectrally resolved lifetime imaging techniques can acquire multiparametric information about the biophysical and biochemical state of the metabolites, these data are evaluated at the whole-cell level, thus providing only limited insights in the activation of metabolic networks at the microscale. To overcome this issue, here we introduce an artificial intelligence-based analysis that, leveraging the multiparametric content of spectrally resolved lifetime images, allows to detect and classify, through an unsupervised learning approach, metabolic clusters, which are regions having almost uniform metabolic properties. This method contextually detects the cellular mitochondrial turnover and the metabolic activation state of intracellular compartments at the pixel level, described by two functions: the cytosolic activation state (CAF) and the mitochondrial activation state (MAF). This method was applied to investigate metabolic changes elicited in the breast cancer cell line MCF-7 by specific inhibitors of glycolysis and electron transport chain, and by the deregulation of a specific mitochondrial enzyme (ACO2) leading to defective aerobic metabolism associated with tumor growth. In this model, mitochondrial fraction undergoes to a 13% increase upon ACO2 overexpression and the MAF function changes abruptly by altering the metabolic state of about the 25% of the mitochondrial pixels.