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Results for “single cell integration”

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PubMed1994

Mechanisms involved in living systems organisation, especially the programming necessary to enable the construction of individuals in three dimensions.

The complexity of the organization of living systems escalates by orders of magnitude in the development, from single precursor cells, not only of the three-dimensional structures characterizing each species but also in the variations necessary to accommodate the 1 million or more separate and identifiable species on this planet. Although the genetic information controlling such information is currently considered to reside in cellular DNA, it is also held that such information is restricted to a linear form encoding specifically for protein. However, this not only fails to explain the co-ordination of the vast number of processes occurring in simple, single cell, organisms, but also the integration of cellular activities to serve the interest of the total system. In particular how can a homeobox containing only genes encoding specifically for proteins organize and implement the mechanisms necessary for three-dimensional development? This, and the organization and implementation of the massive amount of information necessary to execute the construction of such a wide range of species, each in its own unique and exquisite detail, calls for internal programming of a highly complex and sophisticated nature. It is proposed here that such a central computer-analog program does exist, housed in the molecular electronic structure of cellular nucleic acid, primarily in DNA: its possible nature is discussed. Since precursor cells contain only of the order of 10(-10) g of DNA, this proposal involves an increase in information storage efficiency comparable with that already achieved by silicon microprocessors over mechanical calculators.

Animals↗

PubMed2005

Tensile properties of single stress fibers isolated from cultured vascular smooth muscle cells.

Stress fibers (SFs), a contractile bundle of actin filaments, play a critical role in mechanotransduction in adherent cells; yet, the mechanical properties of SFs are poorly understood. Here, we measured tensile properties of single SFs by in vitro manipulation with cantilevers. SFs were isolated from cultured vascular smooth muscle cells with a combination of low ionic-strength extraction and detergent extraction and were stretched until breaking. The breaking force and the Young's modulus (assuming that SFs were isotropic) were, on average, 377 nN and 1.45 MPa, which were approximately 600-fold greater and three orders of magnitude lower, respectively, than those of actin filaments reported previously. Strain-induced stiffening was observed in the force-strain curve. We also found that the extracted SFs shortened to approximately 80% of the original length in an ATP-independent manner after they were dislodged from the substrate, suggesting that SFs had preexisting strain in the cytoplasm. The force required for stretching the single SFs from the zero-stress length back to the original length was approximately 10 nN, which was comparable with the traction force level applied by adherent cells at single adhesion sites to maintain cell integrity. These results suggest that SFs can bear intracellular stresses that may affect overall cell mechanical properties and will impact interpretation of intracellular stress distribution and force-transmission mechanism in adherent cells.

Actinin↗

PubMed2024

Proteomic Heterogeneity of the Extracellular Matrix Identifies Histologic Subtype-Specific Fibroblast in Gastric Cancer.

Gastric cancer (GC) is a highly heterogeneous disease regarding histologic features, genotypes, and molecular phenotypes. Here, we investigate extracellular matrix (ECM)-centric analysis, examining its association with histologic subtypes and patient prognosis in human GC. We performed quantitative proteomic analysis of decellularized GC tissues that characterizes tumorous ECM, highlighting proteomic heterogeneity in ECM components. We identified 20 tumor-enriched proteins including four glycoproteins, serpin family H member 1 (SERPINH1), annexin family (ANXA3/4/5/13), S100A family (S100A6/8/9), MMP14, and other matrisome-associated proteins. In addition, histopathological characteristics of GC reveals differential expression in ECM composition, with the poorly cohesive carcinoma-not otherwise specified (PCC-NOS) subtype being distinctly demarcated from other histologic subtypes. Integrating ECM proteomics with single-cell RNA sequencing, we identified crucial molecular markers in the PCC-NOS-specific stroma. PCC-NOS-enriched matrisome proteins and gene expression signatures of adipogenic cancer-associated fibroblasts (CAFadi) are closely linked, both associated with adverse outcomes in GC. Using tumor microarray analysis, we confirmed the CAFadi surface marker, ATP binding cassette subfamily A member 8 (ABCA8), predominantly present in PCC-NOS tumors. Our ECM-focused analysis paves the way for studies to determine their utility as biomarkers for patient stratification, offering valuable insights for linking molecular and histologic features in GC.

Humans↗

PubMed2000

Immortalization of bovine dental papilla cells with simian virus 40 large t antigen.

Primary cultures of dental papilla-derived cells have a limited lifespan in vitro and can be maintained only up to passage 7-9 before showing senescence, but in vitro investigations often require a large number of cells showing phenotypic characteristics of the original tissue. To overcome this shortcoming, second-passage cells established from calf molar tooth germs by enzymatic pretreatment of the dental papilla were transfected by electroporation with pSV3neo, coding for the oncogene simian virus 40 large t antigen and a neomycin-resistance gene. Under selection by G418 (neomycin), four cell clones were isolated by single cell dilution at passage 15. Integration of simian virus 40 large t antigen and expression of the gene products were determined in cell clones by polymerase chain reaction (PCR) and immunohistochemistry. Four transfected cell lines (clones B, C, D and no. 12) were maintained in culture for over 1.5 years. For cell characterization, gene expression of procollagen alpha1 (I) and osteocalcin was evaluated by reverse transcriptase (RT)-PCR with cDNA obtained from the established cell lines at passage 20. Expression of collagen type I, osteocalcin and dentine phosphoprotein was evaluated immunohistochemically at passage 20 and after 1.5 years of continuous cell culture. Gene expression and the expression of mineralized tissue-specific proteins was demonstrated with RT-PCR and immunohistochemistry within all four immortalized cell lines. Expression of dentine phosphoprotein was observed in three simian virus 40 large t antigen-transfected cell lines, suggesting the immortalization of odontoblast-like cells in vitro. Thus, transfection of bovine dental papilla-derived cells resulted in immortal cell lines exhibiting phenotypic characteristics of the original tissue.

Animals↗

PubMed1996

Scaffold/matrix-attached regions act upon transcription in a context-dependent manner.

Scaffold/matrix-attached regions (S/MARs) are cis-acting elements with a function outside transcribed regions and in introns. Although they usually augment transcriptional rates, their action is highly context-dependent. We cloned an 800 bp S/MAR element from the upstream border of the human interferon-beta domain at various positions within a transcribed region of 4.3 kb. By use of retroviral gene transfer, the vector could be integrated into target cells as a single copy enabling a rigorous definition of the distance between the S/MAR and the transcriptional start site. At a distance of about 4 kb, the S/MAR supported transcriptional initiation, whereas at distances below 2.5 kb, transcription was essentially shut off. Controls proved the functionally of all constructs in the transient expression phase and ruled out any influence of S/MAR position on transcript stability. Moreover, no pausing or premature termination was observed within these elements. We suggest that the protein binding partners of S/MARs change according to the topological status, explaining these divergent S/MAR effects.

3T3 Cells↗

PubMed2026

Multiancestry genome-wide association and multiomics analyses elucidate spatiocellular features of multiple sclerosis genetics.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by demyelination disseminated in space and time. Here we performed a genome-wide association study (GWAS) using 688 MS cases and 205,199 controls from the Japanese population and identified significant associations in the major histocompatibility complex region and a population-specific risk variant in 11q24. Through cross-population GWAS meta-analyses using a total of 29,374 cases and 1,843,563 controls from 4 ancestral populations, we identified 22 novel susceptibility loci. Integration of GWAS and single-cell and single-nucleus RNA sequencing of peripheral blood mononuclear cells and subcortical lesions from patients with MS revealed enrichment of genetic risk factors for MS in CD4+ T helper cell lineage and regulatory T cells, as well as in endothelial cells. Furthermore, spatial transcriptomics of subcortical lesions demonstrated spatial and temporal heterogeneity in associations with MS genetic risk. Our study demonstrates the value of investigation of spatiocellular features of disease genetics across diverse populations and omics modalities.

Humans↗

PubMed2005

Activated IGF-1R inhibits hyperglycemia-induced DNA damage and promotes DNA repair by homologous recombination.

The IGF-1R is a genetic determinant of oxidative stress and longevity. Hyperglycemia induces an exponential increase in the production of a key danger signal, reactive oxygen intermediates, which target genomic DNA. Here, we report for the first time that ligand activation of the IGF-1R prevents hyperglycemia-induced genotoxic stress and enhances DNA repair, maintaining genomic integrity and cell viability. We performed single gel electrophoresis (comet assay) to evaluate DNA damage in serum-starved SV40 murine mesangial cells (MMC) and normal human mesangial cells (NHMC), maintained at high ambient glucose concentration. Hyperglycemia inflicted an impressive array of DNA damage in the form of single-strand breaks (SSBs) and double-strand breaks (DSBs). The inclusion of IGF-1 to culture media of MMC and NHMC prevented hyperglycemia-induced DNA damage. To determine whether DNA damage was mediated by reactive oxygen species (ROS), ROS generation was evaluated, in the presence of IGF-1, or the free radical scavenger n-acetyl-cysteine (NAC). IGF-1 and NAC inhibited hyperglycemic-induced ROS production and hyperglycemia-induced DNA damage. We next asked whether IGF-1 promotes the repair of DSB under hyperglycemic conditions, by homologous recombination (HRR) or nonhomologous end joining (NHEJ). Repair of DSB by NHEJ and HRR was operative in MMC maintained under hyperglycemic conditions. IGF-1 increased HRR by nearly twofold, whereas IGF-1 did not affect DNA repair by NHEJ. IGF-1R enhancement of HRR correlated with the translocation of Rad51 to foci of DNA damage. Inhibition of Rad51 expression by short interfering RNA experiments markedly decreased percentage of MMC positive for Rad51 nuclear foci and increased hyperglycemic DNA damage. We conclude that the activated IGF-1R rescues mesangial cells from hyperglycemia-induced danger signals that target genomic DNA by suppressing ROS and enhancing DNA repair by HRR.

Animals↗

PubMed2025

Molecular hallmarks of excitatory and inhibitory neuronal resilience to Alzheimer's disease.

BACKGROUND: A significant proportion of individuals maintain cognition despite extensive Alzheimer's disease (AD) pathology, known as cognitive resilience. Understanding the molecular mechanisms that protect these individuals could reveal therapeutic targets for AD. METHODS: This study defines molecular and cellular signatures of cognitive resilience by integrating bulk RNA and single-cell transcriptomic data with genetics across multiple brain regions. We analyzed data from the Religious Order Study and the Rush Memory and Aging Project (ROSMAP), including bulk RNA sequencing (n = 631 individuals) and multiregional single-nucleus RNA sequencing (n = 48 individuals). Subjects were categorized into AD, resilient, and control based on β-amyloid and tau pathology, and cognitive status. We identified and prioritized protected cell populations using whole-genome sequencing-derived genetic variants, transcriptomic profiling, and cellular composition. RESULTS: Transcriptomics and polygenic risk analysis position resilience as an intermediate AD state. Only GFAP and KLF4 expression distinguished resilience from controls at tissue level, whereas differential expression of genes involved in nucleic acid metabolism and signaling differentiated AD and resilient brains. At the cellular level, resilience was characterized by broad downregulation of LINGO1 expression and reorganization of chaperone pathways, specifically downregulation of Hsp90 and upregulation of Hsp40, Hsp70, and Hsp110 families in excitatory neurons. MEF2C, ATP8B1, and RELN emerged as key markers of resilient neurons. Excitatory neuronal subtypes in the entorhinal cortex (ATP8B+ and MEF2Chigh) exhibited unique resilience signaling through activation of neurotrophin (BDNF-NTRK2, modulated by LINGO1) and angiopoietin (ANGPT2-TEK) pathways. MEF2C+ inhibitory neurons were over-represented in resilient brains, and the expression of genes associated with rare genetic variants revealed vulnerable somatostatin (SST) cortical interneurons that survive in AD resilience. The maintenance of excitatory-inhibitory balance emerges as a key characteristic of resilience. CONCLUSIONS: We have defined molecular and cellular hallmarks of cognitive resilience, an intermediate state in the AD continuum. Resilience mechanisms include preserved neuronal function, balanced network activity, and activation of neurotrophic survival signaling. Specific excitatory neuronal populations appear to play a central role in mediating cognitive resilience, while a subset of vulnerable interneurons likely provides compensation against AD-associated hyperexcitability. This study offers a framework to leverage natural protective mechanisms to mitigate neurodegeneration and preserve cognition in AD.

Humans↗

PubMed2026

Genome-wide association and multi-omics functional screens reveal the genetic architecture of foveal development.

Foveal hypoplasia causes visual impairment across congenital eye disorders, yet the genetic programmes governing foveal development remain poorly characterised and no tractable model exists for foveal disease. In the first genome-wide association study of foveal hypoplasia, we identified 42 sentinel variants mapping to 54 effector genes supported by ≥ 2 criteria from a variant-to-gene framework incorporating developmental multi-omics. Disruption of six effector genes using mutant lines and CRISPR knockouts in the zebrafish high acuity zone recapitulates structural, functional, and ultrastructural hallmarks of foveal hypoplasia, establishing the first vertebrate disease model. Integration with human foetal single-cell and spatial transcriptomics reveals two temporal waves of effector gene expression and identifies Müller glia as critical mediators of foveal patterning. Phenome-wide analyses reveal foveal variants are pleiotropic with refractive, lenticular, and metabolic traits, connecting foveal development to anterior segment and systemic disease biology. These findings should inform mechanistic studies of macular disease.

Journal Article↗

PubMed2000

Apoptosis and cytokine release induced by ionizing or ultraviolet B radiation in primary and immortalized human keratinocytes.

We have compared the induction of apoptosis and cytokine release by UVB and gamma-radiation in primary (untransformed) and in two immortalized human epithelial/keratinocyte cell lines, HaCaT and KB (KB is now known to be a subline of the ubiquitous keratin-forming tumour cell line HeLa and we therefore designate it HeLa-KB). In both the primary and the immortalized cell lines apoptosis and release of the inflammatory cytokine interleukin-6 are induced rapidly following UVB irradiation. In contrast, only the immortalized cells undergo apoptosis and release interleukin-6 after gamma-irradiation and here the onset of apoptosis and cytokine release are delayed. The same distinction between primary and immortalized cells was observed when double-strand breaks were induced with the anticancer drug mitoxantrone, which stabilizes topoisomerase II-cleavable complexes. We suggest that immortalization may sensitize keratinocytes to the apoptogenic effect of ionizing radiation or mitoxantrone by deregulating normal cell cycle checkpoints. In both human keratinocytes and fibroblasts, cell killing, as assayed by loss of colony-forming ability, is not coupled to apoptosis. Immortalization increases resistance to gamma-radiation killing but sensitizes to apoptosis. In contrast, although immortalization also sensitizes to UVB-induced apoptosis, it does not affect UVB-induced cell killing. Apoptosis unambiguously indicates death at the single cell level but clonal cell survival integrates all the cellular and genetic processes which prevent or permit a scorable clone to develop.

Apoptosis↗

PubMed2005

Evaluation of tension in actin bundle of endothelial cells based on preexisting strain and tensile properties measurements.

Actin bundles in vascular endothelial cells (ECs) play a critical role in transmitting intracellular forces between separate focal adhesion sites. However, quantitative descriptions of tension level in single actin bundles in a physiological condition are still poorly studied. Here, we evaluated magnitude of preexisting tension in a single actin bundle of ECs on the basis of measurements of its preexisting stretching strain and tensile properties. Cultured ECs expressing fluorescently-labeled actin were treated with detergents to extract acin bundles. One end of an actin bundle was then dislodged from the substrate by using a microneedle, resulting in a shortening of the actin bundle due to a release of preexisting tension. Assuming the shortened actin bundle reached its non-stress state, preexisting stretching strain was determined to be 0.24 on average. A tensile test of the dislodged single acin bundle was conducted with a pair of cantilevers to measure the force required for stretching it up to the original length, yielding an estimate of preexisting tension in the actin bundle. The magnitude of the preexisting tension, 4 nN on average, was comparable to previously reported data of the traction force generated by adherent cells at single adhesion sites to keep cell integrity. The Young's modulus of the isolated actin bundle was estimated to be approximately 300 kPa from the tensile tests together with evaluation of average diameter of the isolated actin bundle based on transmission electron microscopy. These data will contribute to better understanding of intracellular stress transmission mechanism in ECs.

Actins↗

PubMed1982

Kinetics of reactions within single erythrocytes: studies by microspectrophotometry.

Single cell spectroscopy has been applied to red blood cells along three different lines of research: a) distribution of single hemoglobin components among different erythrocytes, b) chemical reaction with ligands and diffusion in the red blood cell c) dynamics of hemoglobin S aggregation in single red blood cell. The results reported here show, in general, the great possibilities of using single-cell spectroscopy in investigating complex integrated systems.

Anemia, Sickle Cell↗

PubMed1981

Blood rheology and physiology of microcirculation.

During the last decade, the cellular and plasmatic factors as well as fluid-dynamic conditions responsible for the 'anomalous' rheological behavior of blood have been clarified. Hypotheses about the hemodynamic significance of the hemorheological factors in microvascular perfusion can be formulated and have now been subjected to in vivo tests. Red cells have bipotential flow properties: as a consequence of passive, fluid-drop-like adaptation of RBC and membrane 'tank-treading' rapidly flowing mammalian blood has properties akin to an emulsion with extremely high fluidity - while slowly flowing blood becomes aggregated and assumes the properties of a concentrated reticulated suspension of low fluidity. In addition, in the normal microcirculation the fluidity of blood is maintained by rheological and vasomotor factors despite the fact that the cells are larger than the capillaries. Here, fluidity of blood flow thence critically depends a. on the presence of a minimum of shear forces sufficient to disperse aggregates and deform single cell, b. on the biochemical integrity of the vascular content which is prerequisite for normal RBC deformability, c. on the factors that keep microvascular hematocrit low. Under abnormal biophysical and/or biochemical conditions slowly flowing blood may suffer local loss of fluidity, under conditions of fluidity being abolished without coagulation. In hypoperfused microvascular beds, the variability of flow conditions, as well as the resulting variability of flow behavior is submitted as the cause of non-homogeneous perfusion ('collateral blood viscidation'). Hemorheological active drugs (such as pentoxifylline) cannot improve the deformability (fluidity) of normal erythrocytes, but they can protect the cells against the effect of rigidifying metabolites such as lactate, pH-drop, hyperosmolarity etc. Thus, they can maintain blood fluidity and thence residual flow despite abnormal flow behavior and flow conditions in hypoperfused organs. In vivo results corroborate the theory that factors which increase macroscopic blood viscosity in vitro are of small significance in the normal circulation (where they are likely to be compensated by vasodilatation). However, these same factors are critical to perfusion in the abnormal or decompensated circulation in the absence of adequate flow forces and following the recruitment of the limited vasodilator reserve of vital organs.

Animals↗

PubMed1993

A Burkitt lymphoma cell line with integrated Epstein-Barr virus at a stable chromosome modification site.

Fluorescence in situ hybridization (FISH), Southern, and slot blotting were used to detect Epstein-Barr virus (EBV) DNA and RNA sequences in a Burkitt's lymphoma (BL) cell line derived from a North American patient (NAB-2). FISH analysis after hybridization with a BamHI "V" region of EBV showed that NAB-2 cells have EBV genome integrated at a single site on the short arm of chromosome 2(p13). Single hybridization signals were detected at homologous sites on both chromatoids and nuclei. Furthermore, hybridization of intact nuclei without formamide denaturation and heat allowed the detection of single specific viral RNA transcripts visible as "tracks" or "traces." Southern blot analysis confirmed the integration of EBV genome into the host DNA. Quantification of slot blot hybridization revealed that NAB-2 cells have on average one copy of EBV per cell. Virus insertion into chromosomal DNA caused a stable modification site expressed as a distinctive achromatic region adjacent to the band 2p13. The chromatid lesion at the site of EBV integration involving a recombinogenic and fragile site may have contributed to the development of the NAB-2 BL.

Blotting, Southern↗

PubMed2005

Three GnRH receptor types in laser-captured single cells of the cichlid pituitary display cellular and functional heterogeneity.

The role of multiple gonadotropin-releasing hormone receptor (GnRH-R) types in the regulation of gonadotropic and nongonadotropic cells remains speculative. To address this issue, we developed a technology integrating laser-captured microdissection of single digoxigenin-labeled pituitary cells coupled with real-time quantitative PCR to examine the expression profiles of three endogenous GnRH-R types (R1, R2, and R3) in immature and mature males of tilapia Oreochromis niloticus. Here, in addition to gonadotropes (luteinizing and folicle-stimulating hormone, FSH), we show GnRH-Rs are also present in lactotropes, somatotropes, thyrotropes, melanotropes (melanocyte-stimulating hormone, MSH), corticotropes and somatolactin cells. Subpopulations of pituitary cells express single (42.9%), multiple (32.4%) or lack (24.7%) GnRH-Rs. In immature males, the percentage of FSH cells containing combinations of GnRH-Rs was significantly higher (R1+R2: 24%, P <0.05; R1+R2+R3: 25%, P <0.01) than in mature males, whereas the percentage showing only R1 and R1 and R3 transcripts (P <0.05) was higher in mature males. Significantly greater copies of R1 and R3 transcripts were found in MSH cells of immature and mature males, respectively (P <0.05). GnRH-R transcripts in other pituitary cells (lactotropes, R1 and R2; somatolactin cells/thyrotropes/corticotropes, R1, R2, and R3) were significantly higher in mature males (P <0.05) but were unaltered in somatotropes and luteinizing hormone cells. Thus, FSH and MSH cells are required for both reproductive states, whereas other pituitary cells are recruited only during testicular maturation. The differential expression of GnRH-Rs in gonadotropic and nongonadotropic cells demonstrates cellular and functional heterogeneity of mechanisms controlling normal sexual development.

Animals↗

PubMedDate not supplied

Integrated photothermal flow cytometry in vivo.

The capability of integrated flow cytometry to detect, in real time, moving cells in their natural states in vivo is demonstrated in a study of circulating red and white blood cells in lymph and blood flow of rat mesentery. This system combines dual pump-probe photothermal (PT) techniques, such as PT imaging, the PT thermolens method, and PT velocimetry, with high-resolution (up to 0.3 microm), high-speed (up to 1000 fps) transmission digital microscopy (TDM) and fluorescence imaging. All PT techniques are based on irradiation of cells in rat mesenteric microvessels with a spectrally tunable laser pulse (420 to 570 nm, 8 ns, 0.1 to 300 microJ) and on detection of temperature-dependent variations of the refractive index with a second continuous probe laser beam (633 nm, 1.4 mW). We focus on intravital monitoring of the integral PT response from single, moving, unlabeled cells (from 100 to 500 cells in one measurement). Potential in vivo applications of this new optical tool, called PT flow cytometry (PTFC), are discussed, including identification of selected cells with differences in natural absorptive properties and sizes, determination of laser-induced cell damage, estimation of flow velocity, and monitoring of circulating cells labeled with PT probes.

Animals↗

PubMed1996

Mutagenesis of AS52 cells by low concentrations of lead(II) and mercury(II)

Little is known at the molecular level concerning the genotoxic effects following the acute exposure of eukaryotic cells to low concentrations of lead (II) or mercury (II). There have been conflicting reports concerning the mutagenic potential of these heavy metals, and there have not been any studies performed to determine the molecular mechanism(s) by which these metals are mutagenic. The Chinese hamster ovary cell line, AS52, contains a stably integrated single functional copy of the Escherichia coli xanthine-guanine phosphoribosyltransferase (gpt) gene. Mutations in the gpt gene confer resistance to 6-thioguanine (TG). There was little effect on viability, as measured by relative cloning efficiency, of AS52 cells exposed to lead (II) or mercury (II) up to concentrations of 0.5 microM and 0.3 microM, respectively. However, higher concentrations of the metals caused a significant increase in cell death. There was also a dose-dependent increase in the isolation of mutants resistant to TG in treated cells when compared to non-treated controls. Concentrations of the metals as low as 0.1 microM caused a significant increase in the number of mutants resistant to TG when compared to the number of spontaneous mutants obtained in nontreated controls. While the molecular mechanism(s) by which lead and mercury (II) are genotoxic is unknown, the results of this study demonstrate that low concentrations of lead (II) and mercury (II) are mutagenic in eukaryotic cells.

Animals↗

PubMed2026

A machine learning-derived and functionally validated circadian rhythm signature predicts clinical outcomes and in silico drug sensitivity in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) displays considerable heterogeneity in clinical outcomes, highlighting the need for reliable prognostic biomarkers. While the aberrant expression of circadian rhythm-related genes has been implicated in cancer pathogenesis, its comprehensive role in CRC progression and predicted therapeutic vulnerabilities remains inadequately characterized. METHODS: Bulk and single-cell RNA-sequencing data were integrated from multiple CRC cohorts. A circadian rhythm signature (CRS) was developed through machine learning algorithms and validated for prognostic value. Comprehensive analyses of tumor microenvironment, genomic alterations, and drug sensitivity were performed. Furthermore, the biological function of the core gene, BHLHE40, was validated in CRC cell lines through CCK-8, EdU, and wound healing assays. RESULTS: Single-cell analysis demonstrated an elevated expression signature of circadian rhythm-related genes in dendritic cells. The optimized CRS, comprising 14 circadian rhythm-related genes, successfully categorized patients into high- and low-risk groups. Patients with a high CRS showed markedly poorer overall survival and computationally inferred immunosuppressive features, including reduced CD8+ T cell infiltration and increased M2 macrophage polarization. Genomic analysis revealed enhanced mutation burden in TP53 and alterations in RTK-RAS/WNT pathways. Notably, in vitro assays confirmed that BHLHE40 is significantly overexpressed in CRC cells. Knockdown of BHLHE40 markedly inhibited tumor cell proliferation and migration. Drug sensitivity profiling identified bexarotene and SMER-3 as potential therapeutic options for high-CRS patients. A nomogram integrating CRS with clinical parameters demonstrated superior predictive accuracy for 1-, 3-, and 5-year survival. CONCLUSIONS: The CRS represents a promising prognostic biomarker that reflects tumor immune status and genomic features, providing valuable insights for personalized treatment strategies in CRC.

Circadian rhythm↗