Detection of lipid efflux from foam cell models using a label-free infrared method.

Cardiovascular diseases are still among the leading causes of mortality and morbidity worldwide. The build-up of fatty plaques in the arteries, leading to atherosclerosis, is the most common cause of cardiovascular diseases. The central player in atherosclerotic plaque formation is the foam cell. Foam cells are formed when monocytes infiltrate from the blood stream into the sub-endothelial space, differentiating into macrophages. With the subsequent uptake and storage of lipoprotein, especially low-density lipoprotein (LDL), they change their phenotype to lipid laden cells. Lowering circulating LDL levels, or initiating cholesterol efflux/reverse cholesterol transport in foam cells, is one of the current clinical therapies. Prescription of the pleiotropic drugs, statins, is the most successful therapy for the treatment and prevention of atherosclerosis. In this study, we used a foam cell model from the macrophage cell line, RAW 246.7, and applied the label-free Fourier Transform Infrared Spectroscopy (FTIR) method, i.e. synchrotron-based microFTIR spectroscopy, to study the lipid efflux process initiated by statins in a dose and time dependent manner. We used glass coverslips as substrates for IR analysis. The optical images (visible and fluorescent light) clearly identify the localization and lipid distribution within the foam cells, and the associated changes before and after culturing them with atorvastatin at concentrations of 0.6, 6 and 60 µg mL-1, for a culture duration between 24 to 72 hours. MicroFTIR spectroscopic spectra uniquely displayed the reduction of lipid content, with higher lipid efflux observed at higher doses of, and longer incubation time with, atorvastatin. Principal Component Analysis (PCA) and t-distributed Stochastic Neighbor Embedding (t-SNE) analysis demonstrated defined cluster separation at both lipid (3000-2800 cm-1) and fingerprint (1800-1350 cm-1) regions, with more profound discrimination for the atorvastatin dose treatment than time treatment. The data indicate that combining synchrotron-based microFTIR spectroscopy and using glass substrates for foam cells can offer an alternative tool in atherosclerosis investigation at a molecular level, and through cell morphology.

Optical Photothermal Infrared Microspectroscopy Discriminates for the First Time Different Types of Lung Cells on Histopathology Glass Slides.

The debate of whether a glass substrate can be used in Fourier transform infrared spectroscopy is strongly linked to its potential clinical application. Histopathology glass slides of 1 mm thickness absorb the mid-IR spectrum in the rich fingerprint spectral region. Thus, it is important to assess whether emerging IR techniques can be employed to study biological samples placed on glass substrates. For this purpose, we used optical photothermal infrared (O-PTIR) spectroscopy to study for the first time malignant and non-malignant lung cells with the purpose of identifying IR spectral differences between these cells placed on standard pathology glass slides. The data in this feasibility study showed that O-PTIR can be used to obtain good-quality IR spectra from cells from both the lipid region (3000-2700 cm-1) and the fingerprint region between 1770 and 950 cm-1 but with glass contributions from 1350 to 950 cm-1. A new single-unit dual-range (C-H/FP) quantum cascade laser (QCL) IR pump source was applied for the first time, delivering a clear synergistic benefit to the classification results. Furthermore, O-PTIR is able to distinguish between lung cancer cells and non-malignant lung cells both in the lipid and fingerprint regions. However, when these two spectral ranges are combined, classification accuracies are enhanced with Random Forest modeling classification accuracy results ranging from 96 to 99% across all three studied cell lines. The methodology described here for the first time with a single-unit dual-range QCL for O-PTIR on glass is another step toward its clinical application in pathology.

Clinical applications of infrared and Raman spectroscopy: state of play and future challenges.

Vibrational spectroscopies, based on infrared absorption and/or Raman scattering provide a detailed fingerprint of a material, based on the chemical content. Diagnostic and prognostic tools based on these technologies have the potential to revolutionise our clinical systems leading to improved patient outcome, more efficient public services and significant economic savings. However, despite these strong drivers, there are many fundamental scientific and technological challenges which have limited the implementation of this technology in the clinical arena, although recent years have seen significant progress in addressing these challenges. This review examines (i) the state of the art of clinical applications of infrared absorption and Raman spectroscopy, and (ii) the outstanding challenges, and progress towards translation, highlighting specific examples in the areas of in vivo, ex vivo and in vitro applications. In addition, the requirements of instrumentation suitable for use in the clinic, strategies for pre-processing and statistical analysis in clinical spectroscopy and data sharing protocols, will be discussed. Emerging consensus recommendations are presented, and the future perspectives of the field are assessed, particularly in the context of national and international collaborative research initiatives, such as the UK EPSRC Clinical Infrared and Raman Spectroscopy Network, the EU COST Action Raman4Clinics, and the International Society for Clinical Spectroscopy.

Correction: Clinical applications of infrared and Raman spectroscopy: state of play and future challenges.

Correction for 'Clinical applications of infrared and Raman spectroscopy: state of play and future challenges' by Matthew J. Baker, et al., Analyst, 2018, DOI: 10.1039/c7an01871a.

Evaluation of peroxidative stress of cancer cells in vitro by real-time quantification of volatile aldehydes in culture headspace.

RATIONALE: Peroxidation of lipids in cellular membranes results in the release of volatile organic compounds (VOCs), including saturated aldehydes. The real-time quantification of trace VOCs produced by cancer cells during peroxidative stress presents a new challenge to non-invasive clinical diagnostics, which as described here, we have met with some success. METHODS: A combination of selected ion flow tube mass spectrometry (SIFT-MS), a technique that allows rapid, reliable quantification of VOCs in humid air and liquid headspace, and electrochemistry to generate reactive oxygen species (ROS) in vitro has been used. Thus, VOCs present in the headspace of CALU-1 cancer cell line cultures exposed to ROS have been monitored and quantified in real time using SIFT-MS. RESULTS: The CALU-1 lung cancer cells were cultured in 3D collagen to mimic in vivo tissue. Real-time SIFT-MS analyses focused on the volatile aldehydes: propanal, butanal, pentanal, hexanal, heptanal and malondialdehyde (propanedial), that are expected to be products of cellular membrane peroxidation. All six aldehydes were identified in the culture headspace, each reaching peak concentrations during the time of exposure to ROS and eventually reducing as the reactants were depleted in the culture. Pentanal and hexanal were the most abundant, reaching concentrations of a few hundred parts-per-billion by volume, ppbv, in the culture headspace. CONCLUSIONS: The results of these experiments demonstrate that peroxidation of cancer cells in vitro can be monitored and evaluated by direct real-time analysis of the volatile aldehydes produced. The combination of adopted methodology potentially has value for the study of other types of VOCs that may be produced by cellular damage.

Study of the biochemical effects induced by X-ray irradiations in combination with gadolinium nanoparticles in F98 glioma cells: first FTIR studies at the Emira laboratory of the SESAME synchrotron.

One strategy to improve the clinical outcome of radiotherapy is to use nanoparticles as radiosensitizers. Along this line, numerous studies have shown the enhanced effectiveness of tumour cell killing when nanoparticles are exposed to irradiation. However, the mechanisms of action are not clear yet. In addition to the damage due to a possible local radiation dose enhancement, the interaction of nanoparticles with essential biological macromolecules could lead to changes in the cells, such as cell arrest at radiosensitive phases. Within this framework, vibrational spectroscopy was used to investigate the biochemical changes in F98 glioma cells induced by X-ray irradiations combined with gadolinium nanoparticles. Fourier transform infrared (FTIR) microspectroscopy experiments were performed at the Emira laboratory of the SESAME synchrotron (Jordan), allowing the characterisation of spectral signatures of nanoparticle-induced effects in glioma cells. Multivariate analysis of the spectra recorded using principal component analysis reveals clear differences in the DNA, protein and lipid regions in the presence of nanoparticles. Prior to irradiation, results show that nanoparticles induce biochemical modifications in the cells, probably due to changes in the cellular function. Biochemical alterations are amplified in the presence of radiation. In particular, variations in the intensity and in the position of the PO2(-) symmetric and asymmetric modes are observed due to radiation damage to the DNA, which is increased in nanoparticle-treated cells. At 24 hours post-irradiation, biochemical changes related to the hallmark characteristics of cell death are detected. This includes a shift towards low wavenumbers in the amide I and II bands, relative amplitude changes in the CH2 and CH3 stretching modes, along with DNA chromatin condensation indications. Results were confirmed by two complementary cell viability assays.

A microscopic and macroscopic study of aging collagen on its molecular structure, mechanical properties, and cellular response.

During aging, collagen structure changes, detrimentally affecting tissues' biophysical and biomechanical properties due to an accumulation of advanced glycation end-products (AGEs). In this investigation, we conducted a parallel study of microscopic and macroscopic properties of different-aged collagens from newborn to 2-yr-old rats, to examine the effect of aging on fibrillogenesis, mechanical and contractile properties of reconstituted hydrogels from these collagens seeded with or without fibroblasts. In addition to fibrillogenesis of collagen under the conventional conditions, some fibrillogenesis was conducted alongside a 12-T magnetic field, and gelation rate and AGE content were measured. A nondestructive indentation technique and optical coherence tomography were used to determine the elastic modulus and dimensional changes, respectively. It was revealed that in comparison to younger specimens, older collagens exhibited higher viscosity, faster gelation rates, and a higher AGE-specific fluorescence. Exceptionally, only young collagens formed highly aligned fibrils under magnetic fields. The youngest collagen demonstrated a higher elastic modulus and contraction in comparison to the older collagen. We conclude that aging changes collagen monomer structure, which considerably affects the fibrillogenesis process, the architecture of the resulting collagen fibers and the global network, and the macroscopic properties of the formed constructs.

Spectropathology for the next generation: quo vadis?

Although the potential of vibrational spectroscopy for biomedical applications has been well demonstrated, translation into clinical practice has been relatively slow. This Editorial assesses the challenges facing the field and the potential way forward. While many technological challenges have been addressed to date, considerable effort is still required to gain acceptance of the techniques among the medical community, standardise protocols, extend to a clinically relevant scale, and ultimately assess the health economics underlying clinical deployment. National and international research networks can contribute much to technology development and standardisation. Ultimately, large-scale funding is required to engage in clinical trials and instrument development.

Study of gemcitabine-sensitive/resistant cancer cells by cell cloning and synchrotron FTIR microspectroscopy.

Over the last few years, significant scientific insight on the effects of chemotherapy drugs at cellular level using synchrotron-based FTIR (S-FTIR) microspectroscopy has been obtained. The work carried out so far has identified spectral differences in cancer cells before and after the addition of drugs. However, this had to account for the following issues. First, chemotherapy agents cause both chemical and morphological changes in cells, the latter being responsible for changes in the spectral profile not correlated with biochemical characteristics. Second, as the work has been carried out in mixed populations of cells (resistant and sensitive), it is important to distinguish the spectral differences which are due to sensitivity/resistance to those due to cell morphology and/or cell mixture. Here, we successfully cloned resistant and sensitive lung cancer cells to a chemotherapy drug. This allowed us to study a more uniform population and, more important, allowed us to study sensitive and resistant cells prior to the addition of the drug with S-FTIR microscopy. Principal component analysis (PCA) did not detect major differences in resistant cells prior to and after adding the drug. However, PCA separated sensitive cells prior to and after the addition of the drug. This would indicate that the spectral differences between cells prior to and after adding a drug might reside on those more or less sensitive cells that have been able to remain alive when they were collected to be studied with S-FTIR microspectroscopy. This is a proof of concept and a feasibility study showing a methodology that opens a new way to identify the effects of drugs on more homogeneous cell populations using vibrational spectroscopy.

Quantification by SIFT-MS of acetaldehyde released by lung cells in a 3D model.

Our previous studies have shown that both lung cancer cells and non-malignant lung cells release acetaldehyde in vitro. However, data from other laboratories have produced conflicting results. Furthermore, all these studies have been carried out in 2D models which are less physiological cell growth systems when compared to 3D models. Therefore, we have carried out further work on the release of acetaldehyde by lung cells in 3D collagen hydrogels. Lung cancer cells CALU-1 and non-malignant lung cells NL20 were seeded in these hydrogels at different cell concentrations and the release of acetaldehyde was measured with the Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) technique. The data obtained showed that the amount of acetaldehyde released by both cell types grown in a 3D model is higher when compared to that of the same cells grown in 2D models. More importantly, acetaldehyde from the headspace of lung cancer cells could be measured even at a low cell concentration (10(5) cells per hydrogel). The differential of acetaldehyde release could be, depending on the cell concentration, more than 3 fold higher for cancer cells when compared to non-malignant lung cells. This pilot study is the first to study acetaldehyde emission from albeit only two cell types cultured in 3D scaffolds. Clearly, from such limited data the behaviour of other cell types and of tumour cells in vivo cannot be predicted with confidence. Nevertheless, this work represents another step in the search for volatile biomarkers of tumour cells, the ultimate goal of which is to exploit volatile compounds in exhaled breath and other biological fluids as biomarkers of tumours in vivo.

Identification of different subsets of lung cells using Raman microspectroscopy and whole cell nucleus isolation.

Raman spectroscopy has been widely used to study its possible clinical application in cancer diagnosis. However, in order to make it into clinical practice, it is important that this technique is able not only to identify cancer cells from their normal counterparts, but also from the array of cells present in human tissues. To this purpose, we used Raman spectroscopy to assess whether this technique was able to differentiate not only between lung cancer cells and lung epithelial cells but also from lung fibroblasts. Furthermore, we studied whether the differences were due to cell lineage (epithelial versus fibroblast) or to different proliferative characteristics of cells, and where in the cell compartment these differences might reside. To answer these questions we studied cell cytoplasm, cell nucleus and isolated whole cell nuclei. Our data suggests that Raman spectroscopy can differentiate between lung cancer, lung epithelial cells and lung fibroblasts. More important, it can also differentiate between 2 cells from the same lineage (fibroblast) but with one of them rendered immortal and with an increased proliferative activity. Finally, it seems that the main spectral differences reside in the cell nucleus and that the study of isolated nuclei strengthens the differences between cells.

Domes and semi-capsules as model systems for infrared microspectroscopy of biological cells.

It is well known that infrared microscopy of micrometer sized samples suffers from strong scattering distortions, attributed to Mie scattering. The state-of-the-art preprocessing technique for modelling and removing Mie scattering features from infrared absorbance spectra of biological samples is built on a meta model for perfect spheres. However, non-spherical cell shapes are the norm rather than the exception, and it is therefore highly relevant to evaluate the validity of this preprocessing technique for deformed spherical systems. Addressing these cases, we investigate both numerically and experimentally the absorbance spectra of 3D-printed individual domes, rows of up to five domes, two domes with varying distance, and semi-capsules of varying lengths as model systems of deformed individual cells and small cell clusters. We find that coupling effects between individual domes are small, corroborating previous related literature results for spheres. Further, we point out and illustrate with examples that, while optical reciprocity guarantees the same extinction efficiency for top vs. bottom illumination, a scatterer's internal field may be vastly different in these two situations. Finally, we demonstrate that the ME-EMSC model for preprocessing infrared spectra from spherical biological systems is valid also for deformed spherical systems.

Fourier Transform Infrared microspectroscopy identifies single cancer cells in blood. A feasibility study towards liquid biopsy.

The management of cancer patients has markedly improved with the advent of personalised medicine where treatments are given based on tumour antigen expression amongst other. Within this remit, liquid biopsies will no doubt improve this personalised cancer management. Identifying circulating tumour cells in blood allows a better assessment for tumour screening, staging, response to treatment and follow up. However, methods to identify/capture these circulating tumour cells using cancer cells' antigen expression or their physical properties are not robust enough. Thus, a methodology that can identify these circulating tumour cells in blood regardless of the type of tumour is highly needed. Fourier Transform Infrared (FTIR) microspectroscopy, which can separate cells based on their biochemical composition, could be such technique. In this feasibility study, we studied lung cancer cells (squamous cell carcinoma and adenocarcinoma) mixed with peripheral blood mononuclear cells (PBMC). The data obtained shows, for the first time, that FTIR microspectroscopy together with Random Forest classifier is able to identify a single lung cancer cell in blood. This separation was easier when the region of the IR spectra containing lipids and the amide A (2700 to 3500 cm-1) was used. Furthermore, this work was carried out using glass coverslips as substrates that are widely used in pathology departments. This allows further histopathological cell analysis (staining, immunohistochemistry, …) after FTIR spectra are obtained. Hence, although further work is needed using blood samples from patients with cancer, FTIR microspectroscopy could become another tool to be used in liquid biopsies for the identification of circulating tumour cells, and in the personalised management of cancer.

Morphological analysis of vibrational hyperspectral imaging data.

This study demonstrates the use of standard morphological image processing techniques to reduce the hyperspectral image data of samples, containing discrete particles or domains, to a single average spectrum per particle. The processing is automated and successful even when the particles are in contact. Focal Plane Array, Fourier transform infrared (FTIR) absorbance images of biological cells are used as an example dataset. The large number of spectra in the image (~40,000) can be intelligently averaged to ~100 mean spectra, approximately one per cell, greatly simplifying further analysis. As well as reducing the data, the morphological analysis provides useful information, such as the size of each cell, and allows every spectrum associated with each cell to be identified and analysed independently of the full dataset. Using these methods, combined with principal components analysis, consistent spectral differences are found between the spectra of the whole cells and a cell region approximately corresponding to the nucleus. These spectral differences compare well with previous IR measurements on whole CALU-1 cells and their isolated nuclei, but with a simpler sample preparation. The algorithm created to analyse the CALU-1 cells has been applied to a second cell line (NL20), which has a very different growth morphology, to demonstrate that this processing method is applicable to varied samples with little or no modification.

Raman imaging of single living cells: probing effects of non-cytotoxic doses of an anti-cancer drug.

Identifying cell response to a chemotherapy drug treatment, in particular at the single cell level, is an important issue in patient management. This study aims at evaluating the effect of gemcitabine on single living cells using micro-Raman imaging. We used as a model the non-small lung cancer cell line, Calu-1, exposed to cytostatic doses (1 nM to 1 µM for 24 h and 48 h) of gemcitabine, an antitumor drug currently used in the treatment of lung cancer. Following drug treatment as a function of doses and incubation times, the Raman maps of single living cells were acquired. Cell biomolecules (DNA, RNA, and proteins) were chemically extracted and their spectral signatures used to evaluate their respective distribution in the cellular spectral information of control and treated cells. The quantification of these distributions reveals a significant effect of 100 nM gemcitabine at 48 h incubation (concomitant decrease of nucleic acids and increase of proteins). PCA analyses performed both on nuclear and extracted biomolecules spectra show a time-dependent effect of the drug. These promising results reveal that effects of subtoxic doses can be monitored at the single cell level highlighting the importance of such studies for clinical applications.

Optimization of Sample Preparation Using Glass Slides for Spectral Pathology.

The clinical translation of Fourier transform infrared (FT-IR) microspectroscopy in pathology will require bringing this technique as close as possible to standard practice in pathology departments. An important step is sample preparation for both FT-IR microspectroscopy and pathology. This should entail minimal disruption of standard clinical practice while achieving good quality FT-IR spectral data. In fact, the recently described possibility of obtaining FT-IR spectra of cells placed on glass substrates brings FT-IR microspectroscopy closer to a clinical application. We have now furthered this work in order to identify two different types of lung cancer cells placed on glass coverslips. Two types of sample preparation which are widely used in pathology, cytospin and smear, have been used. Samples were fixed with either methanol, used in pathology, or formalin (4% paraformaldehyde) used widely in spectroscopy. Fixation with methanol (alcohol-based fixative) removed lipids from cells causing a decrease in intensity of the peaks at 2850 cm-1 and 2920 cm-1. Nevertheless, we show for the first time that using either type of sample preparation and fixation on thin glass coverslips allowed to differentiate between two different types of lung cancer cells using either the lipid region or the fingerprint region ranging from 1800 cm-1 to 1350 cm-1. We believe that formalin-fixed cytospin samples would be preferred to study cells on thin coverslips using FT-IR microspectroscopy. This work presents a clear indication for future advances in clinical assessment of samples within pathology units to gain a deeper understanding of cells/tissues under investigation.

Synchrotron-based FTIR spectra of stained single cells. Towards a clinical application in pathology.

Over the last few years, FTIR spectroscopy has become a potential analytical method in tissue and cell studies for cancer diagnosis. This has opened a way towards clinical applications such as a tool that would scan samples to assess the presence or absence of malignant cells in biopsies, or as an aid to help pathologists to better characterise those cells that are suspicious but not diagnostic for cancer. The latter application has the problem that in order to assess these cells pathologists would have already dealt with stained samples. Therefore, it is important to understand how staining would affect the spectra of cells. To this purpose, we have conducted this study in order to clarify, first, how haematoxylin and eosin (H&E) and Papanicolau (Pap) stainings affect the spectra of single cells and, second, whether FTIR spectroscopy could differentiate between stained lung cancer cells and their normal counterparts. Furthermore, different cell preparations (cytospin, and smear) used in cytological diagnosis were assessed. Experiments performed using a bright infrared (IR) source (synchrotron) showed that both H&E and Pap staining induced marked changes in the lipid and amide-II band regions. Despite this, FTIR spectroscopy of already stained cells is capable of differentiating between lung cancer cells and their normal counterparts. The clinical applications of this methodology are discussed.