Application of Advanced Non-Linear Spectral Decomposition and Regression Methods for Spectroscopic Analysis of Targeted and Non-Targeted Irradiation Effects in an In-Vitro Model.

Irradiation of the tumour site during treatment for cancer with external-beam ionising radiation results in a complex and dynamic series of effects in both the tumour itself and the normal tissue which surrounds it. The development of a spectral model of the effect of each exposure and interaction mode between these tissues would enable label free assessment of the effect of radiotherapeutic treatment in practice. In this study Fourier transform Infrared microspectroscopic imaging was employed to analyse an in-vitro model of radiotherapeutic treatment for prostate cancer, in which a normal cell line (PNT1A) was exposed to low-dose X-ray radiation from the scattered treatment beam, and also to irradiated cell culture medium (ICCM) from a cancer cell line exposed to a treatment relevant dose (2 Gy). Various exposure modes were studied and reference was made to previously acquired data on cellular survival and DNA double strand break damage. Spectral analysis with manifold methods, linear spectral fitting, non-linear classification and non-linear regression approaches were found to accurately segregate spectra on irradiation type and provide a comprehensive set of spectral markers which differentiate on irradiation mode and cell fate. The study demonstrates that high dose irradiation, low-dose scatter irradiation and radiation-induced bystander exposure (RIBE) signalling each produce differential effects on the cell which are observable through spectroscopic analysis.

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.

Diagnostic Accuracy of Blood-based Biomarkers for Pancreatic Cancer: A Systematic Review and Meta-analysis.

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate below 5%. Carbohydrate antigen 19-9 (CA19-9) is the most commonly used blood-based biomarker for PDAC in current clinical practice, despite having been shown repeatedly to be inaccurate and have poor diagnostic performance. This review aims to assess the reported diagnostic accuracy of all blood-based biomarkers investigated to date in PDAC, by directly comparing individual biomarkers and multi-biomarker panels, both containing CA19-9 and not (novel). A systematic review was conducted in accordance with PRISMA standards in July 2020. Individualized search strategies for three academic databases identified 5,885 studies between the years 1973 and 2020. After two rounds of screening, 250 studies were included. Data were extracted and assessed for bias. A multivariate three-level meta-analysis with subgroup moderators was run in R using AUC values as effect size. On the basis of this model, the pooled AUC value for all multi-biomarker panels (AUC = 0.898; 95% confidence interval (CI): 0.88-0.91) was significantly higher than all single biomarkers (AUC = 0.803; 95% CI: 0.78-0.83; P < 0.0001). The pooled AUC value for CA19-9 alone was significantly lower compared with the multi-biomarker panels containing CA19-9 (P < 0.0001). For the novel biomarkers, the pooled AUC for single biomarkers was also significantly lower compared with multi-biomarker panels (P < 0.0001). Novel biomarkers that have been repeatedly examined across the literature, such as TIMP-1, CEA, and CA125, are highlighted as promising. These results suggest that CA19-9 may be best used as an addition to a panel of biomarkers rather than alone, and that multi-biomarker panels generate the most robust results in blood-based PDAC diagnosis. Significance: In a systematic review and three-level multivariate meta-analysis, it is shown for the first time that blood-based multi-biomarker panels for the diagnosis of PDAC exhibit superior performance in comparison with single biomarkers. CA19-9 is demonstrated to have limited utility alone, and to perform poorly in patient control cohorts of both healthy and benign individuals. Multi-biomarker panels containing CA19-9 produce the best diagnostic performance overall.

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.

Vibrational spectroscopy of liquid biopsies for prostate cancer diagnosis.

BACKGROUND: Screening for prostate cancer with prostate specific antigen and digital rectal examination allows early diagnosis of prostate malignancy but has been associated with poor sensitivity and specificity. There is also a considerable risk of over-diagnosis and over-treatment, which highlights the need for better tools for diagnosis of prostate cancer. This study investigates the potential of high throughput Raman and Fourier Transform Infrared (FTIR) spectroscopy of liquid biopsies for rapid and accurate diagnosis of prostate cancer. METHODS: Blood samples (plasma and lymphocytes) were obtained from healthy control subjects and prostate cancer patients. FTIR and Raman spectra were recorded from plasma samples, while Raman spectra were recorded from the lymphocytes. The acquired spectral data was analysed with various multivariate statistical methods, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and classical least squares (CLS) fitting analysis. RESULTS: Discrimination was observed between the infrared and Raman spectra of plasma and lymphocytes from healthy donors and prostate cancer patients using PCA. In addition, plasma and lymphocytes displayed differentiating signatures in patients exhibiting different Gleason scores. A PLS-DA model was able to discriminate these groups with sensitivity and specificity rates ranging from 90% to 99%. CLS fitting analysis identified key analytes that are involved in the development and progression of prostate cancer. CONCLUSIONS: This technology may have potential as an alternative first stage diagnostic triage for prostate cancer. This technology can be easily adaptable to many other bodily fluids and could be useful for translation of liquid biopsy-based diagnostics into the clinic.

MicroRNA Analysis of ATM-Deficient Cells Indicate PTEN and CCDN1 as Potential Biomarkers of Radiation Response.

Genetic and epigenetic profile changes associated with individual radiation sensitivity are well documented and have led to enhanced understanding of the mechanisms of the radiation-induced DNA damage response. However, the search continues to identify reliable biomarkers of individual radiation sensitivity. Herein, we report on a multi-biomarker approach using traditional cytogenetic biomarkers, DNA damage biomarkers and transcriptional microRNA (miR) biomarkers coupled with their potential gene targets to identify radiosensitivity in ataxia-telangiectasia mutated (ATM)-deficient lymphoblastoid cell lines (LCL); ATM-proficient cell lines were used as controls. Cells were 0.05 and 0.5 Gy irradiated, using a linear accelerator, with sham-irradiated cells as controls. At 1 h postirradiation, cells were fixed for γ-H2AX analysis as a measurement of DNA damage, and cytogenetic analysis using the G2 chromosomal sensitivity assay, G-banding and FISH techniques. RNA was also isolated for genetic profiling by microRNA (miR) and RT-PCR analysis. A panel of 752 miR were analyzed, and potential target genes, phosphatase and tensin homolog (PTEN) and cyclin D1 (CCND1), were measured. The cytogenetic assays revealed that although the control cell line had functional cell cycle checkpoints, the radiosensitivity of the control and AT cell lines were similar. Analysis of DNA damage in all cell lines, including an additional control cell line, showed elevated γ-H2AX levels for only one AT cell line. Of the 752 miR analyzed, eight miR were upregulated, and six miR were downregulated in the AT cells compared to the control. Upregulated miR-152-3p, miR-24-5p and miR-92-15p and all downregulated miR were indicated as modulators of PTEN and CCDN1. Further measurement of both genes validated their potential role as radiation-response biomarkers. The multi-biomarker approach not only revealed potential candidates for radiation response, but provided additional mechanistic insights into the response in AT-deficient cells.

Effect of hemolysis on Fourier transform infrared and Raman spectra of blood plasma.

Hemolysis is a very common phenomenon and is referred as the release of intracellular components from red blood cells to the extracellular fluid. Hemolyzed samples are often rejected in clinics due to the interference of hemoglobin and intracellular components in laboratory measurements. Plasma and serum based vibrational spectroscopy studies are extensively applied to generate spectral biomarkers for various diseases. However, no studies have reported the effect of hemolysis in blood based vibrational spectroscopy studies. This study was undertaken to evaluate the effect of hemolysis on infrared and Raman spectra of blood plasma. In this study, prostate cancer plasma samples (n = 30) were divided into three groups (nonhemolyzed, mildly hemolyzed, and moderately hemolyzed) based on the degree of hemolysis and FTIR and Raman spectra were recorded using high throughput (HT)-FTIR and HT-Raman spectroscopy. Discrimination was observed between the infrared and Raman spectra of nonhemolyzed and hemolyzed plasma samples using principal component analysis. A classical least square fitting analysis showed differences in the weighting of pure components in nonhemolyzed and hemolyzed plasma samples. Therefore, it is worth to consider the changes in spectral features due to hemolysis when comparing the results within and between experiments.

Monitoring Radiotherapeutic Response in Prostate Cancer Patients Using High Throughput FTIR Spectroscopy of Liquid Biopsies.

Radiation therapy (RT) is used to treat approximately 50% of all cancer patients. However, RT causes a wide range of adverse late effects that can affect a patient's quality of life. There are currently no predictive assays in clinical use to identify patients at risk of normal tissue radiation toxicity. This study aimed to investigate the potential of Fourier transform infrared (FTIR) spectroscopy for monitoring radiotherapeutic response. Blood plasma was acquired from 53 prostate cancer patients at five different time points: prior to treatment, after hormone treatment, at the end of radiotherapy, two months post radiotherapy and eight months post radiotherapy. FTIR spectra were recorded from plasma samples at all time points and the data was analysed using MATLAB software. Discrimination was observed between spectra recorded at baseline versus follow up time points, as well as between spectra from patients showing minimal and severe acute and late toxicity using principal component analysis. A partial least squares discriminant analysis model achieved sensitivity and specificity rates ranging from 80% to 99%. This technology may have potential to monitor radiotherapeutic response in prostate cancer patients using non-invasive blood plasma samples and could lead to individualised patient radiotherapy.

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.

Prediction of DNA damage and G2 chromosomal radio-sensitivity ex vivo in peripheral blood mononuclear cells with label-free Raman micro-spectroscopy.

PURPOSE: Liquid biopsies are a potentially rich store of biochemical information that can be linked to an individual's response to therapeutic treatments, including radiotherapy, and which may ultimately play a role in the individualization of treatment regimens. Peripheral blood mononuclear cells (PBMCs) can be used not only for the biochemical profiling of the individual, but also, being living cells, can provide insights into the individuals response to ionizing radiation exposure. MATERIALS AND METHODS: The present study attempts to link the biochemical profile of lymphocytes within PBMCs obtained through Raman spectroscopy to in vitro measures of low-dose (<0.5Gy) DNA damage response and cytogenetic metrics of radiosensitivity in a cohort of healthy controls and prostate cancer patients (from CTRIAL-IE(ICORG) 08-17, NCT00951535). All parallel metrics to the Raman spectra of the cells were obtained ex vivo in cycling peripheral blood lymphocytes, with radiosensitivity estimated using the G2 chromosomal assay and DNA damage assessed using γH2AX fluorescence. Spectra from a total of 26 healthy volunteers and 22 prostate cancer patients were obtained. RESULTS: The links between both measures of cellular response to ionizing radiation and the Raman spectra were modeled using partial least squares regression (PLSR) and support-vector regression (SVR). It was found that neither regression approach could predict radiation-induced G2 score well, but could predict γH2AX MFI with the SVR outperforming PLSR, implying a non-linear relationship between spectral measurements and measures of DNA damage. CONCLUSIONS: Raman spectroscopy of PBMCs represents a label-free approach for prediction of DNA damage levels for either prospective or retrospective analysis.

Raman spectroscopy for the preoperative diagnosis of thyroid cancer and its subtypes: An in vitro proof-of-concept study.

OBJECTIVE: In 2016, there were an estimated 56 870 new cases of thyroid cancer (TC) in the USA. Fine needle aspiration cytology (FNAC) is the most safe, accurate and cost-effective method for the initial investigation of thyroid nodules. FNAC is limited by the inability to diagnose malignancy in follicular-patterned lesions accurately and, as a result, 20%-30% of cases under investigation for TC are classified as cytologically indeterminate, illustrating a problem with current FNAC procedure. Raman spectroscopy has shown promising results for the detection of many cancers; however, to date there has been no report on the performance of Raman spectroscopy on thyroid cytological samples. The aim of this study was to examine whether Raman spectroscopy could be used to correctly classify cell lines representing benign thyroid cells and various subtypes of TC. METHODS: A benign thyroid cell line and seven TC cell lines were prepared as ThinPrep® cytology slides and analysed with Raman spectroscopy. Principal components analysis and linear discriminant analysis were implemented to develop effective diagnostic algorithms for classification of Raman spectra of different TC subtypes. RESULTS: The spectral differences separating benign and TC cell lines were assigned to differences in the composition of nucleic acids, lipids, carbohydrates and protein in the benign and cancer cells. Good sensitivities (74%-85%), specificities (65%-93%) and diagnostic accuracies (71%-88%) were achieved for the identification of TC. CONCLUSION: These findings suggest that Raman spectroscopy has potential for preoperative TC diagnosis on FNAC samples.

Development of a high throughput (HT) Raman spectroscopy method for rapid screening of liquid blood plasma from prostate cancer patients.

Extensive research has been undertaken on the examination of tissue biopsies using vibrational spectroscopic techniques. However, fewer studies have focused on less invasive and commonly acquired blood samples. Recent studies have shown the ability of Raman and Fourier transform infrared (FTIR) spectroscopy to discriminate between non-cancer controls and cancer cases using blood serum or plasma. Even though many studies have proposed Raman spectroscopy as a potential diagnostic tool in various cancers, the Raman spectroscopic technique has not been introduced as a routine clinical technology. This is due to multiple drawbacks with the application of the technique, including sample preparation, the requirement for expensive substrates and long acquisition times. The current study aims to overcome these limitations and focuses on the translation of Raman spectroscopy into a high throughput clinical diagnostic tool for prostate cancer. In this study, the effect of different instrumental and sample preparation parameters were investigated, with the aim of identifying a combination that would reduce the overall acquisition time for spectra from peripheral blood plasma, reduce the complexity of sample preparation and retain the classification accuracy from Raman spectroscopic diagnostics. A high throughput (HT) system was developed and Raman spectroscopic measurements were performed on plasma samples from 10 prostate cancer patients and 10 healthy volunteers. The spectra were pre-processed and classified by principal component analysis - linear discriminant analysis (PCA-LDA) in the R environment. Statistically significant differences were observed between Raman spectra of prostate cancer patients and non-cancer controls. The (HT) classification resulted in a sensitivity and specificity of 96.5% and 95% respectively. Overall, this study has overcome some of the limitations associated with clinical translation of Raman spectroscopy. The HT-Raman spectroscopy method developed in this study can be used for rapid and accurate diagnosis of prostate cancer using liquid plasma samples.

Raman micro spectroscopy study of the interaction of vincristine with A549 cells supported by expression analysis of bcl-2 protein.

Understanding the interaction of anticancer drugs with model cell lines is important to elucidate the mode of action of these drugs as well as to develop cost effective and rapid screening methods. Raman spectroscopy has been demonstrated to be a valuable technique for high throughput, noninvasive analysis. The interaction of vincristine with a human lung adenocarcinoma cell line (A549) was investigated using Raman micro spectroscopy. The results were correlated with parallel measurements from the MTT cytotoxicity assay, which yielded an IC50 value of 0.10 ± 0.03 µM. The Raman spectral data acquired from vincristine treated A549 cells was analysed to understand its interaction with the nucleus in the cell and elucidate DNA intercalation. The dose dependent spectral changes in the nucleus are analysed by PLS-Jack knifing for the identification of the more significant changes associated with the mode of action of the drug. Results are correlated with a similar dose dependent expression analysis of the bcl-2 protein, an anti-apoptotic protein associated with DNA damage, in the vincristine treated A549 cells using flow cytometry. The results indicate the co-existence of two modes of action, microtubule binding at low doses and DNA intercalation at high doses.

Impaired clearance and enhanced pulmonary inflammatory/fibrotic response to carbon nanotubes in myeloperoxidase-deficient mice.

Advancement of biomedical applications of carbonaceous nanomaterials is hampered by their biopersistence and pro-inflammatory action in vivo. Here, we used myeloperoxidase knockout B6.129X1-MPO (MPO k/o) mice and showed that oxidation and clearance of single walled carbon nanotubes (SWCNT) from the lungs of these animals after pharyngeal aspiration was markedly less effective whereas the inflammatory response was more robust than in wild-type C57Bl/6 mice. Our results provide direct evidence for the participation of MPO - one of the key-orchestrators of inflammatory response - in the in vivo pulmonary oxidative biodegradation of SWCNT and suggest new ways to control the biopersistence of nanomaterials through genetic or pharmacological manipulations.

Comparison of subcellular responses for the evaluation and prediction of the chemotherapeutic response to cisplatin in lung adenocarcinoma using Raman spectroscopy.

Confocal Raman Micro-spectroscopy (CRM) is employed to examine the chemical and physiological effects of anticancer agents, using cisplatin and A549 adenocarcinoma cells as a model compound and test system respectively. Spectral responses of the membrane and cytoplasm of the cell are analysed independently and the results are compared to previously reported spectroscopic studies of the nucleus. Moreover, Raman spectra from the proteins extracted from the control and exposed samples are acquired and analysed to confirm the origin of the molecular changes of the cell membrane and cytoplasm of the A549 cells. Multivariate data analysis techniques including Principal Component Analysis (PCA) and Partial Least Squares Regression (PLSR) along with PLS-Jackknifing are used to analyse the data measured from the cell membrane and cytoplasm of the A549 cells and results are correlated with parallel measurements from the cytotoxicity assay MTT. A PLSR model is used to differentiate between the chemical effect of the chemotherapeutic agent and the physiological response of the A549 cells and to identify regions of the spectrum that are associated with these processes respectively. The PLSR model is also employed to predict, on the basis of the Raman spectra, the effective dose as well as the level of physiological response, using spectra data from the cytoplasmic and cell membrane regions. The effectiveness of the models based on spectral datasets from the cell membrane and cytoplasm is compared to similar models constructed using spectral data from the nuclear region as well as one combining spectral data from all regions. In all cases, higher prediction accuracy is found for regression against the cisplatin dose, and for both regression against the dose and the physiological response, nuclear data yield higher precision.

Correlation of p16(INK4A) expression and HPV copy number with cellular FTIR spectroscopic signatures of cervical cancer cells.

Cervical cancer, a potentially preventable disease, has its main aetiology in infection by high risk human papillomavirus (HR-HPV). Approaches to improving cervical cancer screening and diagnostic methodologies include molecular biological analysis, targeting of biomarker proteins, but also exploration and implementation of new techniques such as vibrational spectroscopy. This study correlates the biomarker protein p16(INK4A) expression levels dependent on HPV copy number with the infrared absorption spectral signatures of the cervical cancer cell lines, HPV negative C33A, HPV-16 positive SiHa and CaSki and HPV-18 positive HeLa. Confocal fluorescence microscopy demonstrated that p16(INK4A) is expressed in all investigated cell lines in both nuclear and cytoplasmic regions, although predominantly in the cytoplasm. Flow cytometry was used to quantify the p16(INK4A) expression levels and demonstrated a correlation, albeit nonlinear, between the reported number of integrated HPV copies and p16(INK4A) expression levels. CaSki cells were found to have the highest level of expression, HeLa intermediate levels, and SiHa and C33A the lowest levels. FTIR spectra revealed differences in nucleic acid, lipid and protein signatures between the cell lines with varying HPV copy number. Peak intensities exhibited increasing tendency in nucleic acid levels and decreasing tendency in lipid levels with increasing HPV copy number, and although they were found to be nonlinearly correlated with the HPV copy number, their dependence on p16(INK4A) levels was found to be close to linear. Principal Component Analysis (PCA) of the infrared absorption spectra revealed differences between nuclear and cytoplasmic spectroscopic signatures for all cell lines, and furthermore clearly differentiated the groups of spectra representing each cell line. Finally, Partial Least Squares (PLS) analysis was employed to construct a model which can predict the p16(INK4A) expression level based on a spectral fingerprint of a cell line, demonstrating the diagnostic potential of spectroscopic techniques.

Evaluation of the potential of Raman microspectroscopy for prediction of chemotherapeutic response to cisplatin in lung adenocarcinoma.

The study of the interaction of anticancer drugs with mammalian cells in vitro is important to elucidate the mechanisms of action of the drug on its biological targets. In this context, Raman spectroscopy is a potential candidate for high throughput, non-invasive analysis. To explore this potential, the interaction of cis-diamminedichloroplatinum(II) (cisplatin) with a human lung adenocarcinoma cell line (A549) was investigated using Raman microspectroscopy. The results were correlated with parallel measurements from the MTT cytotoxicity assay, which yielded an IC(50) value of 1.2 ± 0.2 µM. To further confirm the spectral results, Raman spectra were also acquired from DNA extracted from A549 cells exposed to cisplatin and from unexposed controls. Partial least squares (PLS) multivariate regression and PLS Jackknifing were employed to highlight spectral regions which varied in a statistically significant manner with exposure to cisplatin and with the resultant changes in cellular physiology measured by the MTT assay. The results demonstrate the potential of the cellular Raman spectrum to non-invasively elucidate spectral changes that have their origin either in the biochemical interaction of external agents with the cell or its physiological response, allowing the prediction of the cellular response and the identification of the origin of the chemotherapeutic response at a molecular level in the cell.

Studies of chemical fixation effects in human cell lines using Raman microspectroscopy.

The in vitro study of cellular species using Raman spectroscopy has proven a powerful non-invasive modality for the analysis of cell constituents and processes. This work uses micro-Raman spectroscopy to study the chemical fixation mechanism in three human cell lines (normal skin, normal bronchial epithelium, and lung adenocarcinoma) employing fixatives that preferentially preserve proteins (formalin), and nucleic acids (Carnoy's fixative and methanol-acetic acid). Spectral differences between the mean live cell spectra and fixed cell spectra together with principal components analysis (PCA), and clustering techniques were used to analyse and interpret the spectral changes. The results indicate that fixation in formalin produces spectral content that is closest to that in the live cell and by extension, best preserves the cellular integrity. Nucleic acid degradation, protein denaturation, and lipid leaching were observed with all fixatives and for all cell lines, but to varying degrees. The results presented here suggest that the mechanism of fixation for short fixation times is complex and dependent on both the cell line and fixative employed. Moreover, important spectral changes occur with all fixatives that have consequences for the interpretation of biochemical processes within fixed cells. The study further demonstrates the potential of vibrational spectroscopy in the characterization of complex biochemical processes in cells at a molecular level.