Cancer microcell initiation and determination.

BACKGROUND: Cancer remains one of the leading causes of death worldwide, despite the possibilities to detect early onset of the most common cancer types. The search for the optimal therapy is complicated by the cancer diversity within tumors and the unsynchronized development of cancerous cells. Therefore, it is necessary to characterize cancer cell populations after treatment has been applied, because cancer recurrence is not rare. In our research, we concentrated on small cancer cell subpopulation (microcells) that has a potential to be cancer resistance source. Previously made experiments has shown that these cells in small numbers form in specific circumstances after anticancer treatment. METHODS: In experiments described in this research, the anticancer agents' paclitaxel and doxorubicin were used to stimulate the induction of microcells in fibroblast, cervix adenocarcinoma, and melanoma cell lines. Mainly for the formation of microcells in melanoma cells. The drug-stimulated cells were then characterized in terms of their formation efficiency, morphology, and metabolic activity. RESULTS: We observed the development of cancer microcells and green fluorescent protein (GFP) transfection efficiency after stress. In the time-lapse experiment, we observed microcell formation through a renewal process and GFP expression in the microcells. Additionally, the microcells were viable after anticancer treatment, as indicated by the nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) enzyme activity assay results. Taken together, these findings indicate that cancer microcells are viable and capable of resisting the stress induced by anticancer drugs, and these cells are prone to chemical substance uptake from the environment. CONCLUSION: Microcells are not only common to a specific cancer type, but can be found in any tumor type. This study could help to understand cancer emergence and recurrence. The appearance of microcells in the studied cancer cell population could be an indicator of the individual anticancer therapy effectiveness and patient survival.

CISD3 inhibition drives cystine-deprivation induced ferroptosis.

Ferroptosis, a new form of programmed cell death, not only promotes the pathological process of various human diseases, but also regulates cancer progression. Current perspectives on the underlying mechanisms remain largely unknown. Herein, we report a member of the NEET protein family, CISD3, exerts a regulatory role in cancer progression and ferroptosis both in vivo and in vitro. Pan-cancer analysis from TCGA reveals that expression of CISD3 is generally elevated in various human cancers which are consequently associated with a higher hazard ratio and poorer overall survival. Moreover, knockdown of CISD3 significantly accelerates lipid peroxidation and accentuates free iron accumulation triggered by Xc- inhibition or cystine-deprivation, thus causing ferroptotic cell death. Conversely, ectopic expression of the shRNA-resistant form of CISD3 (CISD3res) efficiently ameliorates the ferroptotic cell death. Mechanistically, CISD3 depletion presents a metabolic reprogramming toward glutaminolysis, which is required for the fuel of mitochondrial oxidative phosphorylation. Both the inhibitors of glutaminolysis and the ETC process were capable of blocking the lipid peroxidation and ferroptotic cell death in the shCISD3 cells. Besides, genetic and pharmacological activation of mitophagy can rescue the CISD3 knockdown-induced ferroptosis by eliminating the damaged mitochondria. Noteworthily, GPX4 acts downstream of CISD3 mediated ferroptosis, which fails to reverse the homeostasis of mitochondria. Collectively, the present work provides novel insights into the regulatory role of CISD3 in ferroptotic cell death and presents a potential target for advanced antitumor activity through ferroptosis.

ZC3HC1 Is a Novel Inherent Component of the Nuclear Basket, Resident in a State of Reciprocal Dependence with TPR.

The nuclear basket (NB) scaffold, a fibrillar structure anchored to the nuclear pore complex (NPC), is regarded as constructed of polypeptides of the coiled-coil dominated protein TPR to which other proteins can bind without contributing to the NB's structural integrity. Here we report vertebrate protein ZC3HC1 as a novel inherent constituent of the NB, common at the nuclear envelopes (NE) of proliferating and non-dividing, terminally differentiated cells of different morphogenetic origin. Formerly described as a protein of other functions, we instead present the NB component ZC3HC1 as a protein required for enabling distinct amounts of TPR to occur NB-appended, with such ZC3HC1-dependency applying to about half the total amount of TPR at the NEs of different somatic cell types. Furthermore, pointing to an NB structure more complex than previously anticipated, we discuss how ZC3HC1 and the ZC3HC1-dependent TPR polypeptides could enlarge the NB's functional repertoire.

Ultrastructure of the Mitochondria-Associated Membranes in Human Tumor Specimens.

Conventional transmission electron microscopy is an essential tool to understand the structure-function relationships and play a vital role in biological research. Mitochondria-associated membranes are linked with cancer processes in a fundamental manner. A conventional transmission electron microscopy method for preparing specimens in clinical and research settings for the study-analysis of the mitochondria-associated membranes in human tumors is presented. The sample processing includes chemical fixation by immersion, dehydration, embedding, polymerization, sectioning, and staining.

Quantitative proteomics identifies the core proteome of exosomes with syntenin-1 as the highest abundant protein and a putative universal biomarker.

Exosomes are extracellular vesicles derived from the endosomal compartment that are potentially involved in intercellular communication. Here, we found that frequently used biomarkers of exosomes are heterogeneous, and do not exhibit universal utility across different cell types. To uncover ubiquitous and abundant proteins, we used an unbiased and quantitative proteomic approach based on super-stable isotope labeling with amino acids in cell culture (super-SILAC), coupled to high-resolution mass spectrometry. In total, 1,212 proteins were quantified in the proteome of exosomes, irrespective of the cellular source or isolation method. A cohort of 22 proteins was universally enriched. Fifteen proteins were consistently depleted in the proteome of exosomes compared to cells. Among the enriched proteins, we identified biogenesis-related proteins, GTPases and membrane proteins, such as CD47 and ITGB1. The cohort of depleted proteins in exosomes was predominantly composed of nuclear proteins. We identified syntenin-1 as a consistently abundant protein in exosomes from different cellular origins. Syntenin-1 is also present in exosomes across different species and biofluids, highlighting its potential use as a putative universal biomarker of exosomes. Our study provides a comprehensive quantitative atlas of core proteins ubiquitous to exosomes that can serve as a resource for the scientific community.

Measuring microenvironment-tuned nuclear stiffness of cancer cells with atomic force microscopy.

Quantification of nuclear stiffness is challenging for cells encapsulated within a 3D extracellular matrix (ECM). Here, we describe an experimental setup for measuring microenvironment-dependent tuning of nuclear stiffness using an atomic force microscope (AFM). In our setup, ECM-coated polyacrylamide hydrogels mimic the stiffness of the microenvironment, enabling the measurement of nuclear stiffness using an AFM probe in live cancer cells. For complete details on the use and execution of this protocol, please refer to Das et al. (2019) (https://doi.org/10.1016/j.matbio.2019.01.001).

Atomic force microscopy to elucidate how peptides disrupt membranes.

Atomic force microscopy is an increasingly attractive tool to study how peptides disrupt membranes. Often performed on reconstituted lipid bilayers, it provides access to time and length scales that allow dynamic investigations with nanometre resolution. Over the last decade, AFM studies have enabled visualisation of membrane disruption mechanisms by antimicrobial or host defence peptides, including peptides that target malignant cells and biofilms. Moreover, the emergence of high-speed modalities of the technique broadens the scope of investigations to antimicrobial kinetics as well as the imaging of peptide action on live cells in real time. This review describes how methodological advances in AFM facilitate new insights into membrane disruption mechanisms.

Laser enhancement of cancer cell destruction by photothermal therapy conjugated glutathione (GSH)-coated small-sized gold nanoparticles.

The current study presents the employment of glutathione (GSH)-modified small-sized gold nanoparticles (AuNPs) ~ 3 nm in photothermal therapy (PTT), to evaluate the targeting and the toxic effect of cancer rather than normal cells. GSH is pH-sensitive surfaces that exhibit a fast response to the variation in pH conditions between normal (~ 7.4) and cancer cells (6-6.5). Results showed a considerable toxic impact via GSH-AuNP accumulation in cancer cells by both green and NIR laser irradiation. A proportional relation of cellular death to AuNP concentration, exposure time, and light-to-heat conversion efficiency has been demonstrated. The small-sized GSH-AuNPs represent promising agents for developing the safety issues of photothermal cancer treatment by the selective targeting of cancer rather than normal cells, reducing the NP toxicity by their size overlapping with the renal clearance barrier of kidney filtration (~ 5.5 nm), and promoting the photothermal performance in the NIR region, in which light penetration into deep cancer regions is more interested.

X-ray microtomography as a new approach for imaging and analysis of tumor spheroids.

Three-dimensional (3D) spheroids mimic important properties of tumors and may soon become a reasonable substitute for animal models and human tissue, eliminating numerous problems related to in vivo and ex vivo experiments and pre-clinical drug trials. Currently, various imaging methods including X-ray microtomography (micro-CT), exist but their spatial resolution is limited. Here, we visualized and provided a morphological analysis of spheroid cell cultures using micro-CT and compared it to that of confocal microscopy. An approach is proposed that can potentially open new diagnostic opportunities to determine the morphology of cancer cells cultured in 3D structures instead of using actual tumors. Spheroids were formed from human melanoma cell lines WM266-4 and WM115 seeded at different cell densities using the hanging drop method. Micro-CT analysis of spheroid showed that spheroid size and shape differed depending on the cell line, initial cell number, and duration of culture. The melanoma cell lines used in this study can successfully be cultured as 3D spheroids and used to substitute human and animal models in pre-clinical studies. The micro-CT allows for high-resolution visualization of the spheroids structure.

A workflow for visualizing human cancer biopsies using large-format electron microscopy.

Recent developments in large format electron microscopy have enabled generation of images that provide detailed ultrastructural information on normal and diseased cells and tissues. Analyses of these images increase our understanding of cellular organization and interactions and disease-related changes therein. In this manuscript, we describe a workflow for two-dimensional (2D) and three-dimensional (3D) imaging, including both optical and scanning electron microscopy (SEM) methods, that allow pathologists and cancer biology researchers to identify areas of interest from human cancer biopsies. The protocols and mounting strategies described in this workflow are compatible with 2D large format EM mapping, 3D focused ion beam-SEM and serial block face-SEM. The flexibility to use diverse imaging technologies available at most academic institutions makes this workflow useful and applicable for most life science samples. Volumetric analysis of the biopsies studied here revealed morphological, organizational and ultrastructural aspects of the tumor cells and surrounding environment that cannot be revealed by conventional 2D EM imaging. Our results indicate that although 2D EM is still an important tool in many areas of diagnostic pathology, 3D images of ultrastructural relationships between both normal and cancerous cells, in combination with their extracellular matrix, enables cancer researchers and pathologists to better understand the progression of the disease and identify potential therapeutic targets.

Cryo-electron microscopy structure and potential enzymatic function of human six-transmembrane epithelial antigen of the prostate 1 (STEAP1).

Six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is an integral membrane protein that is highly up-regulated on the cell surface of several human cancers, making it a promising therapeutic target to manage these diseases. It shares sequence homology with three enzymes (STEAP2-STEAP4) that catalyze the NADPH-dependent reduction of iron(III). However, STEAP1 lacks an intracellular NADPH-binding domain and does not exhibit cellular ferric reductase activity. Thus, both the molecular function of STEAP1 and its role in cancer progression remain elusive. Here, we present a ∼3.0-Šcryo-EM structure of trimeric human STEAP1 bound to three antigen-binding fragments (Fabs) of the clinically used antibody mAb120.545. The structure revealed that STEAP1 adopts a reductase-like conformation and interacts with the Fabs through its extracellular helices. Enzymatic assays in human cells revealed that STEAP1 promotes iron(III) reduction when fused to the intracellular NADPH-binding domain of its family member STEAP4, suggesting that STEAP1 functions as a ferric reductase in STEAP heterotrimers. Our work provides a foundation for deciphering the molecular mechanisms of STEAP1 and may be useful in the design of new therapeutic strategies to target STEAP1 in cancer.

Investigating Tunneling Nanotubes in Cancer Cells: Guidelines for Structural and Functional Studies through Cell Imaging.

By allowing insured communication between cancer cells themselves and with the neighboring stromal cells, tunneling nanotubes (TNTs) are involved in the multistep process of cancer development from tumorigenesis to the treatment resistance. However, despite their critical role in the biology of cancer, the study of the TNTs has been announced challenging due to not only the absence of a specific biomarker but also the fragile and transitory nature of their structure and the fact that they are hovering freely above the substratum. Here, we proposed to review guidelines to follow for studying the structure and functionality of TNTs in tumoral neuroendocrine cells (PC12) and nontumorigenic human bronchial epithelial cells (HBEC-3, H28). In particular, we reported how crucial is it (i) to consider the culture conditions (culture surface, cell density), (ii) to visualize the formation of TNTs in living cells (mechanisms of formation, 3D representation), and (iii) to identify the cytoskeleton components and the associated elements (categories, origin, tip, and formation/transport) in the TNTs. We also focused on the input of high-resolution cell imaging approaches including Stimulated Emission Depletion (STED) nanoscopy, Transmitted and Scanning Electron Microscopies (TEM and SEM). In addition, we underlined the important role of the organelles in the mechanisms of TNT formation and transfer between the cancer cells. Finally, new biological models for the identification of the TNTs between cancer cells and stromal cells (liquid air interface, ex vivo, in vivo) and the clinical considerations will also be discussed.

Nondestructive analysis of tumor-associated membrane protein MUC1 in living cells based on dual-terminal amplification of a DNA ternary complex.

Non-destructive analysis of cells at the molecular level is of critical importance for cell research. At present, immunoassay-based and aptamer-based methods can achieve non-structural destructive cell analysis, but still lead to changes in cells at the molecular level. Here, we have proposed a dual-terminal amplification (DTA) strategy, which enables nondestructive analysis of membrane protein MUC1 without the effect on protein expression and cell viability in living cells. Methods: A fluorophore (Cy5)-labeled DNA ternary complex consisting of three oligonucleotides is designed. It can recognize MUC1 through its aptamer region, and thus make the MUC1 of cells visible under a fluorescence microscope. When DNA polymerase is added, dual-terminal amplification is performed. One direction dissociates aptamer from MUC1, and the other direction, also known as rolling circle amplification (RCA), produces long linear DNA strands, which can be further adopted for quantitative analysis of MUC1. In this way, all reagents are removed from the surface of the cells after the analysis, which allows nondestructive analysis. We named this strategy dual-terminal amplification (DTA) analysis. Results: By using the DTA analysis, both in situ fluorescence imaging analysis and ex situ fluorescence quantitative analysis of MUC1 were achieved. In addition, the aptamer-containing DNA ternary complex stays on cell surface only during the analysis and leaves the cell after the analysis is complete. The cells can be maintained in a non-interfering state for the rest of the time. So after the analysis, it is found that there are no effect on the physiological activity of cells and the expression of target protein even after two rounds of repeatable imaging and quantitative analysis. Conclusion: In summary, we have successfully constructed a strategy for nondestructive analysis of membrane protein in living cells. We believe that this method provides a promising way for the analysis of the key membrane proteins of cells and the versatile utilization of precious cell samples.

Alternative Lengthening of Telomeres: Building Bridges To Connect Chromosome Ends.

Alternative lengthening of telomeres (ALT) is a mechanism of telomere maintenance that is observed in many of the most recalcitrant cancer subtypes. Telomeres in ALT cancer cells exhibit a distinctive nucleoprotein architecture shaped by the mismanagement of chromatin that fosters cycles of DNA damage and replicative stress that activate homology-directed repair (HDR). Mutations in specific chromatin-remodeling factors appear to be key determinants of the emergence and survival of ALT cancer cells. However, these may represent vulnerabilities for the targeted elimination of ALT cancer cells that infiltrate tissues and organs to become devastating tumors. In this review we examine recent findings that provide new insights into the factors and mechanisms that mediate telomere length maintenance and survival of ALT cancer cells.

The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression.

Ferroptosis, a form of regulated cell death caused by lipid peroxidation, was recently identified as a natural tumor suppression mechanism. Here, we show that ionizing radiation (IR) induces ferroptosis in cancer cells. Mechanistically, IR induces not only reactive oxygen species (ROS) but also the expression of ACSL4, a lipid metabolism enzyme required for ferroptosis, resulting in elevated lipid peroxidation and ferroptosis. ACSL4 ablation largely abolishes IR-induced ferroptosis and promotes radioresistance. IR also induces the expression of ferroptosis inhibitors, including SLC7A11 and GPX4, as an adaptive response. IR- or KEAP1 deficiency-induced SLC7A11 expression promotes radioresistance through inhibiting ferroptosis. Inactivating SLC7A11 or GPX4 with ferroptosis inducers (FINs) sensitizes radioresistant cancer cells and xenograft tumors to IR. Furthermore, radiotherapy induces ferroptosis in cancer patients, and increased ferroptosis correlates with better response and longer survival to radiotherapy in cancer patients. Our study reveals a previously unrecognized link between IR and ferroptosis and indicates that further exploration of the combination of radiotherapy and FINs in cancer treatment is warranted.

Identification of a Glass Substrate to Study Cells Using Fourier Transform Infrared Spectroscopy: Are We Closer to Spectral Pathology?

The rising incidence of cancer worldwide is causing an increase in the workload in pathology departments. This, coupled with advanced analysis methodologies, supports a developing need for techniques that could identify the presence of cancer cells in cytology and tissue samples in an objective, fast, and automated way. Fourier transform infrared (FT-IR) microspectroscopy can identify cancer cells in such samples objectively. Thus, it has the potential to become another tool to help pathologists in their daily work. However, one of the main drawbacks is the use of glass substrates by pathologists. Glass absorbs IR radiation, removing important mid-IR spectral data in the fingerprint region (1800 cm-1 to 900 cm-1). In this work, we hypothesized that, using glass coverslips of differing compositions, some regions within the fingerprint area could still be analyzed. We studied three different types of cells (peripheral blood mononuclear cells, a leukemia cell line, and a lung cancer cell line) and lymph node tissue placed on four different types of glass coverslips. The data presented here show that depending of the type of glass substrate used, information within the fingerprint region down to 1350 cm-1 can be obtained. Furthermore, using principal component analysis, separation between the different cell lines was possible using both the lipid region and the fingerprint region between 1800 cm-1 and 1350 cm-1. This work represents a further step towards the application of FT-IR microspectroscopy in histopathology departments.

Radiotherapy dose painting by circadian rhythm based radiomics.

Radiotherapy dose painting is a new dose delivery technique to achieve higher treatment outcome. In this approach, does is escalated to high progressive regions which are heterogeneous and determined by advanced medical imaging. Radiomics is issued as a feasible image quantification method to reveal tumor heterogeneity by extraction of high throughput mineable texture features. On the other hand, circadian rhythm is a given biological process that studied as a critical factor to obtain more effective treatment outcome. In this study, we hypothesized that radiotherapy dose painting could be enhanced by using circadian rhythm that is determined on the radiomics maps obtained from medical images. This hypothesis is based on the idea which circadian rhythm could change the tumor heterogeneity and therefore image features.

Exosome-specific tumor diagnosis via biomedical analysis of exosome-containing microRNA biomarkers.

Exosome-containing microRNAs (exomiRs) can be employed as potential biomarkers for tumor diagnosis and have drawn much attention in the past few years. However, the separation of exosomes and the detection of exomiRs are still inconvenient or even difficult to implement. Thus, it is important to develop a simple, accurate, and reliable strategy for the separation of exosomes and the biomedical analysis of exomiRs. Herein, a novel exosome-specific tumor diagnosis strategy was constructed by integrating the rapid magnetic exosome-enrichment platform and the Ru(bpy)32+-polymer amplified electrochemiluminescence (ECL) strategy. This strategy realized the rapid and efficient capture of tumor-derived exosomes through a biological-affinity identification platform of the EpCAM antibody. The biomedical analysis of exomiRs achieved a preferable specificity and high sensitivity of 103 particles. Furthermore, we investigated the performance index for clinical blood samples from tumor patients; the results indicated that the exosome-specific tumor diagnosis strategy readily and consistently responded to exomiRs. These results indicated that the exosome-specific tumor diagnosis strategy provided new opportunities for the sensitive and efficient analysis of tumor-derived exomiRs. This strategy greatly simplified the biomedical analysis process and established the non-destructive detection mode of fluid biopsy for tumors.

Banding Together: A Systematic Comparison of The Cancer Genome Atlas and the Mitelman Databases.

Cytogenetic aberrations at the single-cell level represent an important characteristic of cancer cells relevant to tumor evolution and prognosis. However, with the advent of The Cancer Genome Atlas (TCGA), there has been a major shift in cancer research to the use of data from aggregate cell populations. Given that tumor cells harbor hundreds to thousands of biologically relevant genetic alterations that manifest as intratumor heterogeneity, these aggregate analyses may miss alterations readily observable at single-cell resolution. Using the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer, we developed an algorithm to parse International System for Cytogenetic Nomenclature notation for quantitative abnormalities. Comparison of the Mitelman database and TCGA demonstrated that the Mitelman database is a powerful resource, and that cytogenetic aberrations captured by traditional approaches used in Mitelman database are on par with population-based genomic analyses used in TCGA. This algorithm will help nonspecialists to overcome the challenges associated with the format and syntax of the Mitelman database. SIGNIFICANCE: A novel in silico approach compares cytogenetic data between the Mitelman database and TCGA, highlighting the advantages and limitations of both datasets.