MiRNA-Mediated Fibrosis in the Out-of-Target Heart following Partial-Body Irradiation.

Recent reports have shown a link between radiation exposure and non-cancer diseases such as radiation-induced heart disease (RIHD). Radiation exposures are often inhomogeneous, and out-of-target effects have been studied in terms of cancer risk, but very few studies have been carried out for non-cancer diseases. Here, the role of miRNAs in the pathogenesis of RIHD was investigated. C57Bl/6J female mice were whole- (WBI) or partial-body-irradiated (PBI) with 2 Gy of X-rays or sham-irradiated (SI). In PBI exposure, the lower third of the mouse body was irradiated, while the upper two-thirds were shielded. From all groups, hearts were collected 15 days or 6 months post-irradiation. The MiRNome analysis at 15 days post-irradiation showed that miRNAs, belonging to the myomiR family, were highly differentially expressed in WBI and PBI mouse hearts compared with SI hearts. Raman spectral data collected 15 days and 6 months post-irradiation showed biochemical differences among SI, WBI and PBI mouse hearts. Fibrosis in WBI and PBI mouse hearts, indicated by the increased deposition of collagen and the overexpression of genes involved in myofibroblast activation, was found 6 months post-irradiation. Using an in vitro co-culture system, involving directly irradiated skeletal muscle and unirradiated ventricular cardiac human cells, we propose the role of miR-1/133a as mediators of the abscopal response, suggesting that miRNA-based strategies could be relevant for limiting tissue-dependent reactions in non-directly irradiated tissues.

Development and Validation of a Raman Spectroscopic Classification Model for Cervical Intraepithelial Neoplasia (CIN).

The mortality associated with cervical cancer can be reduced if detected at the precancer stage, but current methods are limited in terms of subjectivity, cost and time. Optical spectroscopic methods such as Raman spectroscopy can provide a rapid, label-free and nondestructive measurement of the biochemical fingerprint of a cell, tissue or biofluid. Previous studies have shown the potential of Raman spectroscopy for cervical cancer diagnosis, but most were pilot studies with small sample sizes. The aim of this study is to show the clinical utility of Raman spectroscopy for identifying cervical precancer in a large sample set with validation in an independent test set. Liquid-based cervical cytology samples (n = 662) (326 negative, 200 cervical intraepithelial neoplasia (CIN)1 and 136 CIN2+) were obtained as a training set. Raman spectra were recorded from single-cell nuclei and subjected to a partial least squares discriminant analysis (PLSDA). In addition, the PLSDA classification model was validated using a blinded independent test set (n = 69). A classification accuracy of 91.3% was achieved with only six of the blinded samples misclassified. This study showed the potential clinical utility of Raman spectroscopy with a good classification of negative, CIN1 and CIN2+ achieved in an independent test set.

Classification of cytological samples from oral potentially malignant lesions through Raman spectroscopy: A pilot study.

The potential of Raman microspectroscopy of exfoliated cells has been demonstrated for oral cancer diagnosis. In this study, brush biopsies were collected from the buccal mucosa/tongue of healthy donors (n = 31) and from oral mucosal dysplastic lesions (n = 31 patients). Raman spectra were acquired and subjected to partial least squares-discriminant analysis (PLS-DA). The patient samples could be differentiated from healthy donor samples with 96% sensitivity and 95% specificity. Furthermore, PLS-DA models were developed based on cytopathological and histopathological assessment. Low and high grade dysplasia could be discriminated with 64% sensitivity and 65% specificity based on cytopathological assessment, while 81% sensitivity and 86% specificity could be achieved when histopathological assessment was within six months of the brush biopsy sampling. Therefore, this explorative study has successfully demonstrated that Raman spectroscopy may have a role in monitoring patients with dysplasia and may reduce the need for multiple biopsies.

Raman spectral cytopathology for cancer diagnostic applications.

Raman spectroscopy can provide a rapid, label-free, nondestructive measurement of the chemical fingerprint of a sample and has shown potential for cancer screening and diagnosis. Here we report a protocol for Raman microspectroscopic analysis of different exfoliative cytology samples (cervical, oral and lung), covering sample preparation, spectral acquisition, preprocessing and data analysis. The protocol takes 2 h 20 min for sample preparation, measurement and data preprocessing and up to 8 h for a complete analysis. A key feature of the protocol is that it uses the same sample preparation procedure as commonly used in diagnostic cytology laboratories (i.e., liquid-based cytology on glass slides), ensuring compatibility with clinical workflows. Our protocol also covers methods to correct for the spectral contribution of glass and sample pretreatment methods to remove contaminants (such as blood and mucus) that can obscure spectral features in the exfoliated cells and lead to variability. The protocol establishes a standardized clinical routine allowing the collection of highly reproducible data for Raman spectral cytopathology for cancer diagnostic applications for cervical and lung cancer and for monitoring suspicious lesions for oral cancer.

Raman Spectroscopy of Liquid-Based Cervical Smear Samples as a Triage to Stratify Women Who Are HPV-Positive on Screening.

The role of persistent high-risk human papillomavirus (HPV) infection in the development of cervical precancer and cancer is now well accepted, and HPV testing has recently been introduced for primary cervical screening. However, the low specificity of HPV DNA testing can result in large numbers of women with an HPV-positive result, and additional triage approaches are needed to avoid over-referral to colposcopy and overtreatment. The aim of this study was to assess Raman spectroscopy as a potential triage test to discriminate between transient and persistent HPV infection. HPV DNA status and mRNA status were confirmed in ThinPrep® cervical samples (n = 60) using the Cobas 4800 and APTIMA HPV test, respectively. Raman spectra were recorded from single-cell nuclei and subjected to partial least squares discriminant analysis (PLSDA). In addition, the PLSDA classification model was validated using a blinded independent test set (n = 14). Sensitivity of 85% and specificity of 92% were achieved for the classification of transient and persistent HPV infection, and this increased to 90% sensitivity and 100% specificity when mean sample spectra were used instead of individual cellular spectra. This study showed that Raman spectroscopy has potential as a triage test for HPV-positive women to identify persistent HPV infection.

Out-of-Field Hippocampus from Partial-Body Irradiated Mice Displays Changes in Multi-Omics Profile and Defects in Neurogenesis.

The brain undergoes ionizing radiation exposure in many clinical situations, particularly during radiotherapy for brain tumors. The critical role of the hippocampus in the pathogenesis of radiation-induced neurocognitive dysfunction is well recognized. The goal of this study is to test the potential contribution of non-targeted effects in the detrimental response of the hippocampus to irradiation and to elucidate the mechanisms involved. C57Bl/6 mice were whole body (WBI) or partial body (PBI) irradiated with 0.1 or 2.0 Gy of X-rays or sham irradiated. PBI consisted of the exposure of the lower third of the mouse body, whilst the upper two thirds were shielded. Hippocampi were collected 15 days or 6 months post-irradiation and a multi-omics approach was adopted to assess the molecular changes in non-coding RNAs, proteins and metabolic levels, as well as histological changes in the rate of hippocampal neurogenesis. Notably, at 2.0 Gy the pattern of early molecular and histopathological changes induced in the hippocampus at 15 days following PBI were similar in quality and quantity to the effects induced by WBI, thus providing a proof of principle of the existence of out-of-target radiation response in the hippocampus of conventional mice. We detected major alterations in DAG/IP3 and TGF-ß signaling pathways as well as in the expression of proteins involved in the regulation of long-term neuronal synaptic plasticity and synapse organization, coupled with defects in neural stem cells self-renewal in the hippocampal dentate gyrus. However, compared to the persistence of the WBI effects, most of the PBI effects were only transient and tended to decrease at 6 months post-irradiation, indicating important mechanistic difference. On the contrary, at low dose we identified a progressive accumulation of molecular defects that tended to manifest at later post-irradiation times. These data, indicating that both targeted and non-targeted radiation effects might contribute to the pathogenesis of hippocampal radiation-damage, have general implications for human health.

Phenotypic and Functional Characteristics of Exosomes Derived from Irradiated Mouse Organs and Their Role in the Mechanisms Driving Non-Targeted Effects.

Molecular communication between irradiated and unirradiated neighbouring cells initiates radiation-induced bystander effects (RIBE) and out-of-field (abscopal) effects which are both an example of the non-targeted effects (NTE) of ionising radiation (IR). Exosomes are small membrane vesicles of endosomal origin and newly identified mediators of NTE. Although exosome-mediated changes are well documented in radiation therapy and oncology, there is a lack of knowledge regarding the role of exosomes derived from inside and outside the radiation field in the early and delayed induction of NTE following IR. Therefore, here we investigated the changes in exosome profile and the role of exosomes as possible molecular signalling mediators of radiation damage. Exosomes derived from organs of whole body irradiated (WBI) or partial body irradiated (PBI) mice after 24 h and 15 days post-irradiation were transferred to recipient mouse embryonic fibroblast (MEF) cells and changes in cellular viability, DNA damage and calcium, reactive oxygen species and nitric oxide signalling were evaluated compared to that of MEF cells treated with exosomes derived from unirradiated mice. Taken together, our results show that whole and partial-body irradiation increases the number of exosomes, instigating changes in exosome-treated MEF cells, depending on the source organ and time after exposure.

Cold Atmospheric Plasma induces accumulation of lysosomes and caspase-independent cell death in U373MG glioblastoma multiforme cells.

Room temperature Cold Atmospheric Plasma (CAP) has shown promising efficacy for the treatment of cancer but the exact mechanisms of action remain unclear. Both apoptosis and necrosis have been implicated as the mode of cell death in various cancer cells. We have previously demonstrated a caspase-independent mechanism of cell death in p53-mutated glioblastoma multiforme (GBM) cells exposed to plasma. The purpose of this study was to elucidate the molecular mechanisms involved in caspase-independent cell death induced by plasma treatment. We demonstrate that plasma induces rapid cell death in GBM cells, independent of caspases. Accumulation of vesicles was observed in plasma treated cells that stained positive with acridine orange. Western immunoblotting confirmed that autophagy is not activated following plasma treatment. Acridine orange intensity correlates closely with the lysosomal marker Lyso TrackerTM Deep Red. Further investigation using isosurface visualisation of confocal imaging confirmed that lysosomal accumulation occurs in plasma treated cells. The accumulation of lysosomes was associated with concomitant cell death following plasma treatment. In conclusion, we observed rapid accumulation of acidic vesicles and cell death following CAP treatment in GBM cells. We found no evidence that either apoptosis or autophagy, however, determined that a rapid accumulation of late stage endosomes/lysosomes precedes membrane permeabilisation, mitochondrial membrane depolarisation and caspase independent cell death.

Correction: Discrimination of breast cancer from benign tumours using Raman spectroscopy.

[This corrects the article DOI: 10.1371/journal.pone.0212376.].

Discrimination of breast cancer from benign tumours using Raman spectroscopy.

Breast cancer is the most common cancer among women worldwide, with an estimated 1.7 million cases and 522,000 deaths in 2012. Breast cancer is diagnosed by histopathological examination of breast biopsy material but this is subjective and relies on morphological changes in the tissue. Raman spectroscopy uses incident radiation to induce vibrations in the molecules of a sample and the scattered radiation can be used to characterise the sample. This technique is rapid and non-destructive and is sensitive to subtle biochemical changes occurring at the molecular level. This allows spectral variations corresponding to disease onset to be detected. The aim of this work was to use Raman spectroscopy to discriminate between benign lesions (fibrocystic, fibroadenoma, intraductal papilloma) and cancer (invasive ductal carcinoma and lobular carcinoma) using formalin fixed paraffin preserved (FFPP) tissue. Haematoxylin and Eosin stained sections from the patient biopsies were marked by a pathologist. Raman maps were recorded from parallel unstained tissue sections. Immunohistochemical staining for estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2/neu) was performed on a further set of parallel sections. Both benign and cancer cases were positive for ER while only the cancer cases were positive for HER2. Significant spectral differences were observed between the benign and cancer cases and the benign cases could be differentiated from the cancer cases with good sensitivity and specificity. This study has shown the potential of Raman spectroscopy as an aid to histopathological diagnosis of breast cancer, in particular in the discrimination between benign and malignant tumours.

The potential of biobanked liquid based cytology samples for cervical cancer screening using Raman spectroscopy.

Patient samples are unique and often irreplaceable. This allows biobanks to be a valuable source of material. The aim of this study was to assess the ability of Raman spectroscopy to screen for histologically confirmed cases of Cervical Intraepithelial neoplasia (CIN) using biobanked liquid based cytology (LBC) samples. Two temperatures for long term storage were assessed; 80°C and -25°C. The utility of Raman spectroscopy for the detection of CIN was compared for fresh LBC samples and biobanked LBC samples. Two groups of samples were used for the study with one group associated with disease (CIN 3) and the other associated with no disease (cytology negative). The data indicates that samples stored at -80°C are not suitable for assessment by Raman spectroscopy due to a lack of cellular material and the presence of cellular debris. However, the technology can be applied to fresh LBC samples and those stored at -25°C and is, moreover, effective in the discrimination of negative samples from those where CIN 3 has been confirmed. Pooled fresh and biobanked samples are also amenable to the technology and achieve a similar sensitivity and specificity for CIN 3. This study demonstrates that cervical cytology samples stored within biobanks at temperatures that preclude cell lysis can act as a useful resource for Raman spectroscopy and will facilitate research and translational studies in this area.

Raman spectroscopic detection of high-grade cervical cytology: Using morphologically normal appearing cells.

This study aims to detect high grade squamous intraepithelial cells (HSIL) by investigating HSIL associated biochemical changes in morphologically normal appearing intermediate and superficial cells using Raman spectroscopy. Raman spectra (n = 755) were measured from intermediate and superficial cells from negative cytology ThinPrep specimens (n = 18) and from morphologically normal appearing intermediate and superficial cells from HSIL cytology ThinPrep specimens (n = 17). The Raman data was subjected to multivariate algorithms including the standard principal component analysis (PCA)-linear discriminant analysis (LDA) and partial least squares discriminant analysis (PLS-DA) together with random subsets cross-validation for discriminating negative cytology from HSIL. The PCA-LDA method yielded sensitivities of 74.9%, 72.8%, and 75.6% and specificities of 89.9%, 81.9%, and 84.5%, for HSIL diagnosis based on the dataset obtained from intermediate, superficial and mixed intermediate/superficial cells, respectively. The PLS-DA method provided improved sensitivities of 95.5%, 95.2% and 96.1% and specificities of 92.7%, 94.7% and 93.5% compared to the PCA-LDA method. The results demonstrate that the biochemical signatures of morphologically normal appearing cells can be used to discriminate between negative and HSIL cytology. In addition, it was found that mixed intermediate and superficial cells could be used for HSIL diagnosis as the biochemical differences between negative and HSIL cytology were greater than the biochemical differences between intermediate and superficial cell types.

Improved removal of blood contamination from ThinPrep cervical cytology samples for Raman spectroscopic analysis.

There is an unmet need for methods to help in the early detection of cervical precancer. Optical spectroscopy-based techniques, such as Raman spectroscopy, have shown great potential for diagnosis of different cancers, including cervical cancer. However, relatively few studies have been carried out on liquid-based cytology (LBC) pap test specimens and confounding factors, such as blood contamination, have been identified. Previous work reported a method to remove blood contamination before Raman spectroscopy by pretreatment of the slides with hydrogen peroxide. The aim of the present study was to extend this work to excessively bloody samples to see if these could be rendered suitable for Raman spectroscopy. LBC ThinPrep specimens were treated by adding hydrogen peroxide directly to the vial before slide preparation. Good quality Raman spectra were recorded from negative and high grade (HG) cytology samples with no blood contamination and with heavy blood contamination. Good classification between negative and HG cytology could be achieved for samples with no blood contamination (sensitivity 92%, specificity 93%) and heavy blood contamination (sensitivity 89%, specificity 88%) with poorer classification when samples were combined (sensitivity 82%, specificity 87%). This study demonstrates for the first time the improved potential of Raman spectroscopy for analysis of ThinPrep specimens regardless of blood contamination.

A study of hormonal effects in cervical smear samples using Raman spectroscopy.

Raman spectroscopy is a powerful tool that has the potential to be used as a screening method for cervical cancer. It is a label-free, low-cost method providing a biochemical fingerprint of a given sample. The objective of this study was to address patient-to-patient variability contributed by hormonal effects due to the menstrual cycle, the use of hormone-based contraceptives (HC) and the onset of menopause, and to determine if these changes would affect the ability to successfully identify dyskaryotic cells. Raman spectra were recorded from unstained ThinPrep cervical samples (45 cytology negative and 15 high-grade dyskaryosis (high-grade squamous intraepithelial lesion, HSIL) samples using a HORIBA Jobin Yvon XploRA system. HPV DNA testing was also performed. Clinical data collected included date of the last menstrual period, the use of HC and/or menopausal status. Spectral changes were observed depending on the day of the menstrual cycle and on the use of HC. Despite this, HSIL could be discriminated from normal cells regardless of the day on which the sample was taken or the use of HC.

Raman spectral signatures of cervical exfoliated cells from liquid-based cytology samples.

It is widely accepted that cervical screening has significantly reduced the incidence of cervical cancer worldwide. The primary screening test for cervical cancer is the Papanicolaou (Pap) test, which has extremely variable specificity and sensitivity. There is an unmet clinical need for methods to aid clinicians in the early detection of cervical precancer. Raman spectroscopy is a label-free objective method that can provide a biochemical fingerprint of a given sample. Compared with studies on infrared spectroscopy, relatively few Raman spectroscopy studies have been carried out to date on cervical cytology. The aim of this study was to define the Raman spectral signatures of cervical exfoliated cells present in liquid-based cytology Pap test specimens and to compare the signature of high-grade dysplastic cells to each of the normal cell types. Raman spectra were recorded from single exfoliated cells and subjected to multivariate statistical analysis. The study demonstrated that Raman spectroscopy can identify biochemical signatures associated with the most common cell types seen in liquid-based cytology samples; superficial, intermediate, and parabasal cells. In addition, biochemical changes associated with high-grade dysplasia could be identified suggesting that Raman spectroscopy could be used to aid current cervical screening tests.

Raman spectroscopy for screening and diagnosis of cervical cancer.

Cervical cancer is the fourth most common cancer in women worldwide and mainly affects younger women. The mortality associated with cervical cancer can be reduced if the disease is detected at the pre-cancer stage. Current best-practice methods include cytopathology, HPV testing, and histopathology, but these methods are limited in terms of subjectivity, cost, and time. There is an unmet clinical need for new methods to aid clinicians in the early detection of cervical pre-cancer. These methods should be objective and rapid and require minimal sample preparation. Raman spectroscopy is a vibrational spectroscopic technique by which incident radiation is used to induce vibrations in the molecules of a sample and the scattered radiation may be used to characterise the sample in a rapid and non-destructive manner. Raman spectroscopy is sensitive to subtle biochemical changes occurring at the molecular level, enabling spectral variations corresponding to disease onset to be detected. Over the past 15 years, there have been numerous reports revealing the potential of Raman spectroscopy together with multivariate statistical analysis for the detection of a variety of cancers. This paper discusses the recent advances and challenges for cervical-cancer screening and diagnosis and offers some perspectives for the future.