Merits and boundaries of the BCLC staging and treatment algorithm: Learning from the past to improve the future with a novel proposal.

In this Expert Opinion, we thoroughly analyse the Barcelona Clinic Liver Cancer (BCLC) staging and treatment algorithm for hepatocellular carcinoma (HCC) that, since 1999, has standardised HCC management, offering a structured approach for the prognostic evaluation and treatment of patients with HCC. The first part of the article presents the strengths and evolutionary improvements of the BCLC staging system. Nevertheless, both patient characteristics and available treatments have changed in the last two decades, limiting the role of the BCLC criteria for treatment allocation in a growing number of patients. As therapeutic options expand and become more effective, the stage-linked treatment decision-making algorithm may lead to undertreatment and suboptimal outcomes for patients with disease beyond early-stage HCC. Consequently, strict adherence to BCLC criteria is limited in expert centres, particularly for patients diagnosed beyond early-stage HCC. Although the BCLC system remains the benchmark against which other therapeutic frameworks must be judged, the era of precision medicine calls for patient-tailored therapeutic decision-making (by a multidisciplinary tumour board) rather than stage-dictated treatment allocation. Acknowledging this conceptual difference in clinical management, the second part of the article describes a novel "multiparametric therapeutic hierarchy", which integrates a comprehensive assessment of clinical factors, biomarkers, technical feasibility, and resource availability. Lastly, considering the increasing efficacy of locoregional and systemic treatments, the concept of "converse therapeutic hierarchy" is introduced. These treatments can increase the feasibility (conversion approach) and effectiveness (adjuvant approach of systemic therapy) of potentially curative approaches to greatly improve clinical outcomes.

Nanoscale Force-Mapping-Based Quantification of Low-Abundance Methylated DNA.

Methylation changes at cytosine-guanine dinucleotide (CpG) sites in genes are closely related to cancer development. Thus, detection and quantification of low-abundance methylated DNA is critical for early diagnosis. Here, we report an atomic force microscopy (AFM)-based quantification method for DNA that contains methyl-CpG at a specific site, without any treatment to the target DNA such as chemical labeling, fluorescence tagging, or amplification. We employed AFM-tip-tethered methyl-CpG-binding proteins to probe surface-captured methylated DNA. We observed a linear correlation (R2 = 0.982) between the input copy number and detected copy number, in the low copy number regime (10 or fewer; subattomolar concentrations). For a mixture of methylated and nonmethylated DNA that resembles clinical samples, we were still able to quantify the methylated DNA. These results highlight the potential of our force-mapping-based quantification method for wide applications in early detection of diseases associated with methylated DNA.

Force Mapping Reveals the Spatial Distribution of Individual Proteins in a Neuron.

Conventional methods for studying the spatial distribution and expression level of proteins within neurons have primarily relied on immunolabeling and/or signal amplification. Here, we present an atomic force microscopy (AFM)-based nanoscale force mapping method, where Anti-LIMK1-tethered AFM probes were used to visualize individual LIMK1 proteins in cultured neurons directly through force measurements. We observed that the number density of LIMK1 decreased in neuronal somas after the cells were depolarized. We also elucidated the spatial distribution of LIMK1 in single spine areas and found that the protein predominantly locates at heads of spines rather than dendritic shafts. The study demonstrates that our method enables unveiling of the abundance and spatial distribution of a protein of interest in neurons without signal amplification or labeling. We expected that this approach should facilitate the studies of protein expression phenomena in depth in a wide range of biological systems.

Modified cytosines versus cytosine in a DNA polymerase: retrieving thermodynamic and kinetic constants at the single molecule level.

DNA methylation plays key roles in various areas, such as gene expression, regulation, epigenetics, and cancers. Since 5-methylcytosine (5mC) is commonly present in methylated DNA, characterizing the binding kinetics and thermodynamics of the nucleotide to the enzymatic pocket can help to understand the DNA replication process. Furthermore, 5-carboxycytosine (5caC) is a form that appears through the iterative oxidation of 5mC, and its effect on the DNA replication process is still not well known. Here, we immobilized a DNA polymerase (DNAP) with an orientation control on a tip of an atomic force microscope (AFM), and observed the interaction between the immobilized deoxyguanosine triphosphate (dGTP) on the surface and the DNAP in the presence of a DNA duplex. The interaction probability increased as the concentration of the DNA strand, and the affinity constant between the DNAP and DNA was obtained by fitting the change. Increasing the concentration of dGTP in solution diminished the interaction probability, and a fitting allowed us to retrieve the affinity constant between dGTP and the DNAP holding the DNA in the reaction pocket. Because the dissociation constant could be obtained through the loading rate dependence of the unbinding force value, both affinity and kinetic constants for cytosine (C), 5mC, and 5caC in the DNAP were compared in the light of the steric and electronic effect of the substituents at 5-position of cytosine.

Nanoscale imaging reveals miRNA-mediated control of functional states of dendritic spines.

Dendritic spines are major loci of excitatory inputs and undergo activity-dependent structural changes that contribute to synaptic plasticity and memory formation. Despite the existence of various classification types of spines, how they arise and which molecular components trigger their structural plasticity remain elusive. microRNAs (miRNAs) have emerged as critical regulators of synapse development and plasticity via their control of gene expression. Brain-specific miR-134s likely regulate the morphological maturation of spines, but their subcellular distributions and functional impacts have rarely been assessed. Here, we exploited atomic force microscopy to visualize in situ miR-134s, which indicated that they are mainly distributed at nearby dendritic shafts and necks of spines. The abundance of miR-134s varied between morphologically and functionally distinct spine types, and their amounts were inversely correlated with their postulated maturation stages. Moreover, spines exhibited reduced contents of miR-134s when selectively stimulated with beads containing brain-derived neurotropic factor (BDNF). Taken together, in situ visualizations of miRNAs provided unprecedented insights into the "inverse synaptic-tagging" roles of miR-134s that are selective to inactive/irrelevant synapses and potentially a molecular means for modifying synaptic connectivity via structural alteration.

Nanoaggregates Derived from Amyloid-beta and Alpha-synuclein Characterized by Sequential Quadruple Force Mapping.

Overlapping of Alzheimer's disease and Parkinson's disease is associated with the formation of hetero-oligomers derived from amyloid-beta and alpha-synuclein. However, the structural identity of the hetero-oligomer has yet to be elucidated, particularly at high resolution. Here, with atomic force microscopy, the surface structure of hetero-oligomer was examined with four AFM tips tethering one of the selected antibodies recognizing N-terminus or C-terminus of each peptide. All aggregates were found to be hetero-oligomers, and probability of recognizing the termini is higher than that for the homo-oligomers, suggesting that the termini of the former have a greater tendency to be located at the surface or the termini have more freedom to be recognized, probably through loose packing. The methodology in this study provides us with a new approach to elucidate the structure of such aggregates at the single-molecule level, allowing the exploration of other intrinsically disordered proteins frequently found in nature.

DNA-Engineerable Ultraflat-Faceted Core-Shell Nanocuboids with Strong, Quantitative Plasmon-Enhanced Fluorescence Signals for Sensitive, Reliable MicroRNA Detection.

There has been enormous interest in understanding and utilizing plasmon-enhanced fluorescence (PEF) with metal nanostructures, but maximizing the enhancement in a reproducible, quantitative manner while reliably controlling the distance between dyes and metal particle surface for practical applications is highly challenging. Here, we designed and synthesized fluorescence-amplified nanocuboids (FANCs) with highly enhanced and controlled PEF signals, and fluorescent silica shell-coated FANCs (FS-FANCs) were then formed to fixate the dye position and increase particle stability and fluorescence signal intensity for biosensing applications. By uniformly modifying fluorescently labeled DNA on Au nanorods and forming ultraflat Ag shells on them, we were able to reliably control the distance between fluorophores and Ag surface and obtained an ∼186 fluorescence enhancement factor with these FANCs. Importantly, FS-FANCs were utilized as fluorescent nanoparticle tags for microarray-based miRNA detection, and we achieved >103-fold higher sensitivity than commercially available chemical fluorophores with 100 aM to 1 pM dynamic range.

Direct Detection of Low Abundance Genes of Single Point Mutation.

Cell-free DNA (cfDNA) analysis, specifically circulating tumor DNA (ctDNA) analysis, provides enormous opportunities for noninvasive early assessment of cancers. To date, PCR-based methods have led this field. However, the limited sensitivity/specificity of PCR-based methods necessitates the search for new methods. Here, we describe a direct approach to detect KRAS G12D mutated genes in clinical ctDNA samples with the utmost LOD and sensitivity/specificity. In this study, MutS protein was immobilized on the tip of an atomic force microscope (AFM), and the protein sensed the mismatched sites of the duplex formed between the capture probe on the surface and mutated DNA. A noteworthy LOD (3 copies, 0.006% allele frequency) was achieved, along with superb sensitivity/specificity (100%/100%). These observations demonstrate that force-based AFM, in combination with the protein found in nature and properly designed capture probes/blockers, represents an exciting new avenue for ctDNA analysis.

Ultra-Sensitive and Label-Free Probing of Binding Affinity Using Recognition Imaging.

Reliable quantification of binding affinity is important in biotechnology and pharmacology and increasingly coupled with a demand for ultrasensitivity, nanoscale resolution, and minute sample amounts. Standard techniques are not able to meet these criteria. This study provides a new platform based on atomic force microscopy (AFM)-derived recognition imaging to determine affinity by visualizing single molecular bindings on nanosize dendrons. Using DNA hybridization as a demonstrator, an AFM sensor adorned with a cognate binding strand senses and localizes target DNAs at nanometer resolution. To overcome the limitations of speed and resolution, the AFM cantilever is sinusoidally oscillated close to resonance conditions at small amplitudes. The equilibrium dissociation constant of capturing DNA duplexes was obtained, yielding 2.4 × 10-10 M. Our label-free single-molecular biochemical analysis approach evidences the utility of recognition imaging and analysis in quantifying biomolecular interactions of just a few hundred molecules.

Binary Structure of Amyloid Beta Oligomers Revealed by Dual Recognition Mapping.

Amyloid beta (Aß) oligomers are widely considered to be the causative agent of Alzheimer's disease (AD), a progressive neurodegenerative disorder. Determining the structure of oligomers is, therefore, important for understanding the disease and developing therapeutic agents; however, elucidating the structure has been proven difficult due to heterogeneity, noncrystallinity, and variability. Herein, we investigated homo- and hetero-oligomers of Aß40 and Aß42 using atomic force microscopy (AFM) and revealed characteristics of the molecular structure. By examining the surface of individual oligomers with sequential N- and C-terminus specific antibody-tethered tips, we simultaneously mapped the N- and C-terminus distributions and the elastic modulus. Interestingly, both the N- and C-termini of Aß peptides were recognized on the oligomer surface, and the termini detected pixel regions exhibited a lower elastic modulus than silent pixel regions. These two types of regions were randomly distributed on the oligomer surface.

Direct Quantification of Trace Amounts of a Chronic Myeloid Leukemia Biomarker Using Locked Nucleic Acid Capture Probes.

Molecular monitoring is indispensable for the clinical management of chronic myeloid leukemia (CML) patients. Real-time quantitative polymerase chain reaction (RT-qPCR) is the gold standard for the quantitative assessment of BCR-ABL transcript levels, which are critical in clinical decision-making. However, the frequent recurrence of the disease after drug discontinuation for 60% of patients has necessitated more sensitive and specific techniques to detect residual BCR-ABL transcripts. Here, we describe a quantification method for the detection of BCR-ABL targets at very low concentrations (<10 copies/sample) in the presence of a million copies of normal BCR and ABL genes. In this method, a fully modified locked nucleic acid (LNA) and a LNA/DNA chimera were used as capture probes, and the quantitative imaging mode of atomic force microscopy (AFM) was employed. Targets with one of the major breakpoints (found in more than 95% of CML patients), b3a2 and b2a2, were quantified. The BCR-ABL target captured on a miniaturized LNA-probe spot was scanned at nanometric resolution, and the samples containing one to ten copies of the BCR-ABL genes were examined. It was observed that the highest sensitivity, i.e., the detection of a single copy of the target gene, could be achieved through multiple runs, and the observed cluster number was well correlative (adjusted R2 = 0.999) to the target copy number in the sample solution. This observation clearly demonstrates that the LNA-based platform is effective in quantifying BCR-ABL targets with extremely low copy numbers, highlighting the potential applicability of AFM for use in the direct quantification of such targets without amplification or labeling.

Force Measurement for the Interaction between Cucurbit[7]uril and Mica and Self-Assembled Monolayer in the Presence of Zn2+ Studied with Atomic Force Microscopy.

Force spectroscopy with atomic force microscopy (AFM) revealed that cucurbit[7]uril (CB[7]) strongly binds to a mica surface in the presence of cations. Indeed, Zn2+ was observed to facilitate the self-assembly of CB[7] on the mica surface, whereas monocations, such as Na+, were less effective. The progression of the process and the cation-mediated self-assembled monolayer were characterized using AFM, and the observed height of the layer agrees well with the calculated CB[7] value (9.1 Å). We utilized force-based AFM to further study the interaction of CB[7] with guest molecules. To this end, CB[7] was immobilized on a glass substrate, and aminomethylferrocene (am-Fc) was conjugated onto an AFM tip. The single-molecule interaction between CB[7] and am-Fc was monitored by collecting the unbinding force curves. The force histogram showed single ruptures and a unimodal distribution, and the most probable unbinding force value was 101 pN in deionized water and 86 pN in phosphate-buffered saline buffer. The results indicate that the unbinding force was larger than that of streptavidin-biotin measured under the same conditions, whereas the dissociation constant was smaller by 1 order of magnitude (0.012 s-1 vs 0.13 s-1). Furthermore, a high-resolution adhesion force map showed a part of the CB[7] cavities on the surface.

Quantification of Fewer than Ten Copies of a DNA Biomarker without Amplification or Labeling.

Polymerase chain reaction (PCR) is a highly sensitive diagnosis technique for detection of nucleic acids and for monitoring residual disease; however, PCR can be unreliable for samples containing very few target molecules. Here, we describe a quantification method, using force-distance (FD) curve based atomic force microscopy (AFM) to detect a target DNA bound to small (1.4-1.9 µm diameter) probe DNA spots, allowing mapping of entire spots to nanometer resolution. Using a synthetic BCR-ABL fusion gene sequence target, we examined samples containing between one and 10 target copies. A high degree of correlation (r(2) = 0.994) between numbers of target copies and detected probe clusters was observed, and the approach could detect the BCR-ABL biomarker when only a single copy was present, although multiple screens were required. Our results clearly demonstrate that FD curve-based imaging is suitable for quantitative analysis of fewer than 10 copies of DNA biomarkers without amplification, modification, or labeling.

Visualization and Quantification of MicroRNA in a Single Cell Using Atomic Force Microscopy.

MicroRNAs (miRNAs) play critical roles in controlling various cellular processes, and the expression levels of individual miRNAs can be considerably altered in pathological conditions such as cancer. Accurate quantification of miRNA at the single-cell level will lead to a better understanding of miRNA function. Here, we present a direct and sensitive method for miRNA detection using atomic force microscopy (AFM). A hybrid binding domain (HBD)-tethered tip enabled mature miRNAs, but not premature miRNAs, to be located individually on an adhesion force map. By scanning several sections of a micrometer-sized DNA spot, we were able to quantify the copy number of miR-134 in a single neuron and demonstrate that the expression was increased upon cell activation. Moreover, we visualized individual miR-134s on fixed neurons after membrane removal and observed 2-4 miR-134s in the area of 1.0 × 1.0 µm(2) of soma. The number increased to 8-14 in stimulated neurons, and this change matches the ensemble-averaged increase in copy number. These findings indicate that miRNAs can be reliably quantified at the single cell level with AFM and that their distribution can be mapped at nanometric lateral resolution without modification or amplification. Furthermore, the analysis of miRNAs, mRNAs, and proteins in the same sample or region by scanning sequentially with different AFM tips would let us accurately understand the post-transcriptional regulation of biological processes.

Reading single DNA with DNA polymerase followed by atomic force microscopy.

The importance of DNA sequencing in the life sciences and personalized medicine is continually increasing. Single-molecule sequencing methods have been developed to analyze DNA directly without the need for amplification. Here, we present a new approach to sequencing single DNA molecules using atomic force microscopy (AFM). In our approach, four surface-conjugated nucleotides were examined sequentially with a DNA polymerase-immobilized AFM tip. By observing the specific rupture events upon examination of a matching nucleotide, we could determine the template base bound in the polymerase's active site. The subsequent incorporation of the complementary base in solution enabled the next base to be read. Additionally, we observed that the DNA polymerase could incorporate the surface-conjugated dGTP when the applied force was controlled by employing the force-clamp mode.

Nanoscale analysis of a functionalized polythiophene surface by adhesion mapping.

Functionalized ethylenedioxythiophene (EDOT) monomers, hydroxymethyl EDOT (EDOT-OH), and zwitterionic phosphorylcholine EDOT (EDOT-PC) were electropolymerized to prepare the homopolymers poly(EDOT-OH) and poly(EDOT-PC), and mixtures of these monomers were used to produce the copolymer poly(EDOT-OH)-co-poly(EDOT-PC). Force-extension-curve-based atomic force microscopy (AFM) was utilized to analyze the surfaces of the films. The PEDOT-OH film yielded force-extension curves for short stretching, and the PEDOT-PC film yielded curves for long stretching. A dendron-modified AFM tip with anthracene groups tethered at the end resulted in adhesion maps with the highest contrast. The analytical data for the copolymer films correlated with the corresponding monomer composition, and the maps revealed that the average size for the copolymer nanodomains ranged from 10-14 nm. This approach can be applied to studies aimed at understanding the surface structure of other relevant polymers and copolymers at the nanoscale level.

Direct quantitative analysis of HCV RNA by atomic force microscopy without labeling or amplification.

Force-based atomic force microscopy (AFM) was used to detect HCV (hepatitis C virus) RNA directly and to quantitatively analyse it without the need for reverse transcription or amplification. Capture and detection DNA probes were designed. The former was spotted onto a substrate with a conventional microarrayer, and the latter was immobilized on an AFM probe. To control the spacing between the immobilized DNAs on the surface, dendron self-assembly was employed. Force-distance curves showed that the mean force of the specific unbinding events was 32 ± 5 pN, and the hydrodynamic distance of the captured RNA was 30-60 nm. Adhesion force maps were generated with criteria including the mean force value, probability of obtaining the specific curves and hydrodynamic distance. The maps for the samples whose concentrations ranged from 0.76 fM to 6.0 fM showed that cluster number has a linear relationship with RNA concentration, while the difference between the observed number and the calculated one increased at low concentrations. Because the detection limit is expected to be enhanced by a factor of 10 000 when a spot of 1 micron diameter is employed, it is believed that HCV RNA of a few copy numbers can be detected by the use of AFM.

AFM Imaging Reveals MicroRNA-132 to be a Positive Regulator of Synaptic Functions.

The modification of synaptic and neural connections in adults, including the formation and removal of synapses, depends on activity-dependent synaptic and structural plasticity. MicroRNAs (miRNAs) play crucial roles in regulating these changes by targeting specific genes and regulating their expression. The fact that somatic and dendritic activity in neurons often occurs asynchronously highlights the need for spatial and dynamic regulation of protein synthesis in specific milieu and cellular loci. MicroRNAs, which can show distinct patterns of enrichment, help to establish the localized distribution of plasticity-related proteins. The recent study using atomic force microscopy (AFM)-based nanoscale imaging reveals that the abundance of miRNA(miR)-134 is inversely correlated with the functional activity of dendritic spine structures. However, the miRNAs that are selectively upregulated in potentiated synapses, and which can thereby support prospective changes in synaptic efficacy, remain largely unknown. Using AFM force imaging, significant increases in miR-132 in the dendritic regions abutting functionally-active spines is discovered. This study provides evidence for miR-132 as a novel positive miRNA regulator residing in dendritic shafts, and also suggests that activity-dependent miRNAs localized in distinct sub-compartments of neurons play bi-directional roles in controlling synaptic transmission and synaptic plasticity.

Mapping of surface-immobilized DNA with force-based atomic force microscopy.

Single-stranded 50-mer, 100-mer, and 150-mer DNAs were immobilized on a surface, and force-based atomic force microscopy (AFM) was employed to examine their behavior. A complementary 20-mer probe DNA on an AFM tip was used for the measurements. High-resolution maps were generated, and relevant parameters, including the force, stretching distance, unbinding probability, cluster size, and degree of distortion, were analyzed. Due to thermal drift, the cluster shape became increasingly distorted as the scan speed was decreased and as the map area was reduced. The cluster radius increased with the number of base (N), and the radius was proportional to N(0.6) (r = 0.977) and N(0.53) (r = 0.991). Due to the effect of the pulling angle, the apparent values of the stretching distance and the unbinding force decreased as the AFM probe was moved away from the center position; these values can be described as a function of sin θ.