Granulocyte colony-stimulating factor promotes an aggressive phenotype of colon and breast cancer cells with biochemical changes investigated by single-cell Raman microspectroscopy and machine learning analysis.

Granulocyte colony-stimulating factor (G-CSF) is produced at high levels in several cancers and is directly linked with metastasis in gastrointestinal (GI) cancers. In order to further understand the alteration of molecular compositions and biochemical features triggered by G-CSF treatment at molecular and cell levels, we sought to investigate the long term treatment of G-CSF on colon and breast cancer cells measured by label-free, non-invasive single-cell Raman microspectroscopy. Raman spectrum captures the molecule-specific spectral signatures ("fingerprints") of different biomolecules presented on cells. In this work, mouse breast cancer line 4T1 and mouse colon cancer line CT26 were treated with G-CSF for 7 weeks and subsequently analyzed by machine learning based Raman spectroscopy and gene/cytokine expression. The principal component analysis (PCA) identified the Raman bands that most significantly changed between the control and G-CSF treated cells. Notably, here we proposed the concept of aggressiveness score, which can be derived from the posterior probability of linear discriminant analysis (LDA), for quantitative spectral analysis of tumorigenic cells. The aggressiveness score was effectively applied to analyze and differentiate the overall cell biochemical changes of G-CSF-treated two model cancer cells. All these tumorigenic progressions suggested by Raman analysis were confirmed by pro-tumorigenic cytokine and gene analysis. A high correlation between gene expression data and Raman spectra highlights that the machine learning based non-invasive Raman spectroscopy offers emerging and powerful tools to better understand the regulation mechanism of cytokines in the tumor microenvironment that could lead to the discovery of new targets for cancer therapy.

Imaging of PD-L1 in single cancer cells by SERS-based hyperspectral analysis.

We developed a hyperspectral imaging tool based on surface-enhanced Raman spectroscopy (SERS) probes to determine the expression level and visualize the distribution of PD-L1 in individual cells. Electron-microscopic analysis of PD-L1 antibody - gold nanorod conjugates demonstrated binding the cell surface and internalization into endosomal vesicles. Stimulation of cells with IFN-γ or metformin was used to confirm the ability of SERS probes to report treatment-induced changes. The multivariate curve resolution-alternating least squares (MCR-ALS) analysis of spectra provided a greater signal-noise ratio than single peak mapping. However, single peak mapping allowed a systematic subtraction of background and the removal of non-specific binding and endocytic SERS signals. The mean or maximum peak height in the cell or the mean peak height in the area of specific PD-L1 positive pixels was used to estimate the PD-L1 expression levels in single cells. The PD-L1 levels were significantly up-regulated by IFN-γ and inhibited by metformin in human lung cancer cells from the A549 cell line. In conclusion, the method of analyzing hyperspectral SERS imaging data together with systematic and comprehensive removal of non-specific signals allows SERS imaging to be a quantitative tool in the detection of the cancer biomarker, PD-L1.

A multi-scale approach to study biochemical and biophysical aspects of resveratrol on diesel exhaust particle-human primary lung cell interaction.

Diesel exhaust particles (DEPs) are major air pollutants that lead to numerous human disorders, especially pulmonary diseases, partly through the induction of oxidative stress. Resveratrol is a polyphenol that ameliorates the production of reactive oxygen species (ROS) and delays aging-related processes. Herein we studied the cytoprotective effect of resveratrol on DEP-exposed human lung cells in a factorial experimental design. This work investigates biophysical features including cellular compositions and biomechanical properties, which were measured at the single-cell level using confocal Raman microspectroscopy (RM) and atomic force microscopy (AFM), respectively. Principal component analysis (PCA), hierarchical cluster analysis (HCA) and partial least square regression (PLS) analysis were applied to analyze Raman spectra with and without resveratrol protection. The health status of individual cells could be effectively predicted using an index derived from characteristic Raman spectral peak (e.g., 1006 cm-1) based on PLS model. AFM measurements indicated that cellular adhesion force was greatly reduced, while Young's modulus was highly elevated in resveratrol treated DEP-exposed cells. Anti-oxidant resveratrol reduced DEP-induced ROS production and suppressed releases of several cytokines and chemokines. These findings suggest resveratrol may enhance resistance of human lung cells (e.g., SAEC) to air pollutants (e.g. DEPs).

Microfluidic chip for non-invasive analysis of tumor cells interaction with anti-cancer drug doxorubicin by AFM and Raman spectroscopy.

Raman spectroscopy has been playing an increasingly significant role for cell classification. Here, we introduce a novel microfluidic chip for non-invasive Raman cell natural fingerprint collection. Traditional Raman spectroscopy measurement of the cells grown in a Polydimethylsiloxane (PDMS) based microfluidic device suffers from the background noise from the substrate materials of PDMS when intended to apply as an in vitro cell assay. To overcome this disadvantage, the current device is designed with a middle layer of PDMS layer sandwiched by two MgF2 slides which minimize the PDMS background signal in Raman measurement. Three cancer cell lines, including a human lung cancer cell A549, and human breast cancer cell lines MDA-MB-231 and MDA-MB-231/BRMS1, were cultured in this microdevice separately for a period of three days to evaluate the biocompatibility of the microfluidic system. In addition, atomic force microscopy (AFM) was used to measure the Young's modulus and adhesion force of cancer cells at single cell level. The AFM results indicated that our microchannel environment did not seem to alter the cell biomechanical properties. The biochemical responses of cancer cells exposed to anti-cancer drug doxorubicin (DOX) up to 24 h were assessed by Raman spectroscopy. Principal component analysis over the Raman spectra indicated that cancer cells untreated and treated with DOX can be distinguished. This PDMS microfluidic device offers a non-invasive and reusable tool for in vitro Raman measurement of living cells, and can be potentially applied for anti-cancer drug screening.

Gd2O3-doped silica @ Au nanoparticles for in vitro imaging cancer biomarkers using surface-enhanced Raman scattering.

There has been an interest in developing multimodal approaches to combine the advantages of individual imaging modalities, as well as to compensate for respective weaknesses. We previously reported a composite nano-system composed of gadolinium-doped mesoporous silica nanoparticle and gold nanoparticle (Gd-Au NPs) as an efficient MRI contrast agent for in vivo cancer imaging. However, MRI lacks sensitivity and is unsuitable for in vitro cancer detection. Thus, here we performed a study to use the Gd-Au NPs for detection and imaging of a widely recognized human cancer biomarker, epidermal growth factor receptor (EGFR), in individual human cancer cells with surface-enhanced Raman scattering (SERS). The Gd-Au NPs were sequentially conjugated with a monoclonal antibody recognizing EGFR and a Raman reporter molecule, 4-meraptobenzoic acid (MBA), to generate a characteristic SERS signal at 1075cm-1. By spatially mapping the SERS intensity at 1075cm-1, cellular distribution of EGFR and its relocalization on the plasma membrane were measured in situ. In addition, the EGFR expression levels in three human cancer cell lines (S18, A431 and A549) were measured using this SERS probe, which were consistent with the comparable measurements using immunoblotting and immunofluorescence. Our SERS results show that functionalized Gd-Au NPs successfully targeted EGFR molecules in three human cancer cell lines and monitored changes in single cell EGFR distribution in situ, demonstrating its potential to study cell activity under physiological conditions. This SERS study, combined with our previous MRI study, suggests the Gd-Au nanocomposite is a promising candidate contrast agent for multimodal cancer imaging.

In vitro detection of diesel exhaust particles induced human lung carcinoma epithelial cells damage and the effect of resveratrol.

People are taking up antioxidants in their daily diet and being exposed to a potential diesel exhaust particles (DEP)-containing environment. Thus it is important to study in vitro cellular responses when cells are exposed to DEP with or without antioxidant treatment. The investigation of DEP and resveratrol (RES) on cellular biophysical and biochemical changes is needed to better understand the mechanisms of DEP and RES in mammalian cells. A combination of two non-invasive techniques (atomic force microscopy, AFM, and Raman spectroscopy, RM) and multimodal tools were applied to evaluate the biophysical, biochemical alterations and cytokine, membrane potential and cell cycle of cells with or without RES pretreatment to different times of DEP exposure. AFM results indicated that RES protected cells from DEP-induced damage to cytoskeleton and cell architectures, and noted that RES treatments also attenuated DEP-induced alterations in cell elasticity and surface adhesion force over DEP incubation time. RM monitored the changes in characteristic Raman peak intensities of DNA and protein over the DEP exposure time for both RES and non-RES treated groups. The cytokine and chemokine changes quantified by Multiplex ELISA revealed that the inflammatory responses were enhanced with the increase in DEP exposure time and that RES enhanced the expression levels of cytokine and chemokine. This work demonstrated that significant biophysical and biochemical changes in cells might be relevant to early pathological changes induced by DEP damage. Copyright © 2016 John Wiley & Sons, Ltd.

In vitro biomechanical properties, fluorescence imaging, surface-enhanced Raman spectroscopy, and photothermal therapy evaluation of luminescent functionalized CaMoO4:Eu@Au hybrid nanorods on human lung adenocarcinoma epithelial cells.

Highly dispersible Eu3+-doped CaMoO4@Au-nanorod hybrid nanoparticles (HNPs) exhibit optical properties, such as plasmon resonances in the near-infrared region at 790 nm and luminescence at 615 nm, offering multimodal capabilities: fluorescence imaging, surface-enhanced Raman spectroscopy (SERS) detection and photothermal therapy (PTT). HNPs were conjugated with a Raman reporter (4-mercaptobenzoic acid), showing a desired SERS signal (enhancement factor 5.0 × 105). The HNPs have a heat conversion efficiency of 25.6%, and a hyperthermia temperature of 42°C could be achieved by adjusting either concentration of HNPs, or laser power, or irradiation time. HNPs were modified with antibody specific to cancer biomarker epidermal growth factor receptor, then applied to human lung cancer (A549) and mouse hepatocyte cells (AML12), and in vitro PTT effect was studied. In addition, the biomechanical properties of A549 cells were quantified using atomic force microscopy. This study shows the potential applications of these HNPs in fluorescence imaging, SERS detection, and PTT with good photostability and biocompatibility.

Near-infrared photothermal therapy of Prussian-blue-functionalized lanthanide-ion-doped inorganic/plasmonic multifunctional nanostructures for the selective targeting of HER2-expressing breast cancer cells.

We report the synthesis, characterization, and application of Prussian blue (PB) functionalized CaMoO4:Eu@SiO2@Au nanorod hybrid nanoparticles (HNPs), with multimodal capabilities such as fluorescence imaging, surface-enhanced Raman spectroscopy (SERS) detection and photothermal therapy (PTT). The average size of CaMoO4:Eu@SiO2 NPs was 206 nm. The HNPs are highly dispersible in water for several weeks without settling and show a strong absorption in the near-infrared region, overlapping with the PB absorption between 600 nm and 1000 nm and the surface plasmon resonance of Au nanorods around 800 nm. Upon 808 nm laser excitation, HNPs show hyperthermia temperature (∼43 °C). Moreover, PB-functionalized NPs can be used in clinical trials for the treatment of radioactive exposure, and PB acts as a Raman reporter molecule (2152 cm-1 characteristic peak) with good biosafety and stability in the human body. In addition, coating the surface of CaMoO4:Eu NPs with both SiO2 and Au nanorods increases the biocompatibility of the HNPs. Furthermore, the PTT efficiency of human epidermal growth factor receptor 2 (HER2) antibody-conjugated HNPs on MDA-MB-435 cancerous cells was significantly higher than that of hepatocyte cells (noncancerous). This is due to the greater uptake of HNPs on cancerous cells than on noncancerous cells. Together, this study shows the potential applications of these HNPs in fluorescence imaging, SERS detection, and PTT functionalities with good photostability and biocompatibility.

Simultaneous topographic and recognition imaging of epidermal growth factor receptor (EGFR) on single human breast cancer cells.

Epidermal growth factor receptor (EGFR) plays an important role in signaling pathway of the development of breast cancer cells. Since EGFR overexpresses in most breast cancer cells, it is regarded as a biomarker molecule of breast cancer cells. Here we demonstrated a new AFM technique-topography and recognition (TREC) imaging-to simultaneously obtain highly sensitive and specific molecular recognition images and high-resolution topographic images of EGFR on single breast cancer cells.

In vitro biophysical, microspectroscopic and cytotoxic evaluation of metastatic and non-metastatic cancer cells in responses to anti-cancer drug.

The Breast Cancer Metastasis Suppressor 1 (BRMS1) is a nucleo-cytoplasmic protein that suppresses cancer metastasis without affecting the growth of the primary tumor. Previous work has shown that it decreases the expression of protein mediators involved in chemoresistance. This study measured the biomechanical and biochemical changes in BRMS1 expression and the responses of BRMS1 to drug treatments on cancer cells in vitro. The results show that BRMS1 expression affects biomechanical properties by decreasing the Young's modulus and adhesion force of breast cancer cells after doxorubicin (DOX) exposure. Raman spectral bands corresponding to DNA/RNA, lipids and proteins were similar for all cells after DOX treatment. The expression of cytokines were similar for cancer cells after DOX exposure, although BRMS1 expression had different effects on the secretion of cytokines for breast cancer cells. The absence of significant changes on apoptosis, reactive oxygen species (ROS) expression and cell viability after BRMS1 expression shows that BRMS1 has little effect on cellular chemoresistance. Analyzing cancer protein expression is critical in evaluating therapeutics. Our study may provide evidence of the benefit of metastatic suppressor expression before chemotherapy.

Imaging of epidermal growth factor receptor on single breast cancer cells using surface-enhanced Raman spectroscopy.

Epidermal growth factor receptor (EGFR) is widely used as a biomarker for pathological grading and therapeutic targeting of human cancers. This study investigates expression, spatial distribution as well as the endocytosis of EGFR in single breast cancer cells using surface-enhanced Raman spectroscopy (SERS). By incubating anti-EGFR antibody conjugated SERS nanoprobes with an EGFR-over-expressing cancer cell line, A431, EGFR localization was measured over time and found to be located primarily at the cell surface. To further validate the constructed SERS probes, we applied this SERS probes to detect the EGFR expression on breast cancer cells (MDA-MB-435, MDA-MB-231) and their counterpart cell lines in which EGFR expression was down-regulated by breast cancer metastasis suppressor 1 (BRMS1). The results showed that SERS method not only confirms immunoblot data measuring EGFR levels, but also adds new insights regarding EGFR localization and internalization in living cells which is impossible in immunoblot method. Thus, SERS provides a powerful new tool to measure biomarkers in living cancer cells.

Non-lithographic patterning of phage-displayed peptides with wrinkled elastomers.

The development of controlled patterning of phage (viruses) could expand opportunities for both fundamental studies and creating various materials platforms. Inducing the elastomeric instability of PDMS film provides a non-lithographic, tuneable, controlled method for generating micro/nanoscale wrinkle patterns. Phage display has emerged as a powerful method for selecting peptides that possess enhanced selectivity and binding affinity toward a variety of targets. In this report, we demonstrate the non-lithographic patterning of phage-displayed peptides with wrinkled elastomers. Our results show that the phage-displayed peptides can be patterned on specific locations in controlled and tuneable ways, be transferred to other substrates and induce the self-assembly of hybrid materials. We anticipate that these results could open up exciting opportunities in fundamental studies and in applications ranging from sensors, hybrid materials, self-assembly, surface and interface, to micro/nanoelectronics.

Sensing biophysical alterations of human lung epithelial cells (A549) in the context of toxicity effects of diesel exhaust particles.

Diesel exhaust particles (DEP) in urban air are associated with numerous respiratory diseases. The role of underlying biomechanics in cytotoxicity of individual lung cells relating to DEP exposure is unclear. In this study, atomic force microscopy (AFM), confocal Raman microspectroscopy (RM), and fluorescence (FL) microscopy were used to monitor alterations of single A549 cells exposed to DEP. Results revealed a significant decrease in membrane surface adhesion force and a significant change in cell elasticity as a function of DEP-cell interaction time, and the dynamic changes in cellular biocomponents which were reflected by changes of characteristic Raman bands: 726 cm(-1) (adenine), 782 cm(-1) (uracil, cytosine, thymine), 788 cm(-1) (O-P-O), 1006 cm(-1) (phenylalanine), and 1320 cm(-1) (guanine) after DEP exposure. These findings suggest that the combination of multi-instruments (e.g., AFM/FL) may offer an exciting platform for investigating the roles of biophysical and biochemical responses to particulate matter-induced cell toxicity.

Subcellular spectroscopic markers, topography and nanomechanics of human lung cancer and breast cancer cells examined by combined confocal Raman microspectroscopy and atomic force microscopy.

The nanostructures and hydrophobic properties of cancer cell membranes are important for membrane fusion and cell adhesion. They are directly related to cancer cell biophysical properties, including aggressive growth and migration. Additionally, chemical component analysis of the cancer cell membrane could potentially be applied in clinical diagnosis of cancer by identification of specific biomarker receptors expressed on cancer cell surfaces. In the present work, a combined Raman microspectroscopy (RM) and atomic force microscopy (AFM) technique was applied to detect the difference in membrane chemical components and nanomechanics of three cancer cell lines: human lung adenocarcinoma epithelial cells (A549), and human breast cancer cells (MDA-MB-435 with and without BRMS1 metastasis suppressor). Raman spectral analysis indicated similar bands between the A549, 435 and 435/BRMS1 including ~720 cm(-1) (guanine band of DNA), 940 cm(-1) (skeletal mode polysaccharide), 1006 cm(-1) (symmetric ring breathing phenylalanine), and 1451 cm(-1) (CH deformation). The membrane surface adhesion forces for these cancer cells were measured by AFM in culture medium: 0.478 ± 0.091 nN for A549 cells, 0.253 ± 0.070 nN for 435 cells, and 1.114 ± 0.281 nN for 435/BRMS1 cells, and the cell spring constant was measured at 2.62 ± 0.682 mN m(-1) for A549 cells, 2.105 ± 0.691 mN m(-1) for 435 cells, and 5.448 ± 1.081 mN m(-1) for 435/BRMS1 cells.

Toxicity effects of short term diesel exhaust particles exposure to human small airway epithelial cells (SAECs) and human lung carcinoma epithelial cells (A549).

In this study, confocal Raman spectroscopy, atomic force microscope (AFM) and multiplex ELISA were applied to analyze the biophysical responses (biomechanics and biospectroscopy) of normal human primary small airway epithelial cells (SAECs) and human lung carcinoma epithelial A549 cells to in vitro short term DEP exposure (up to 2h). Raman spectra revealed the specific cellular biomolecular changes in cells induced by DEP compared to unexposed control cells. Principal component analysis was successfully applied to analyze spectral differences between control and treated groups from multiple individual cells, and indicated that cell nuclei are more sensitive than other cell locations. AFM measurements indicated that 2h of DEP exposure induced a significant decrease in cell elasticity and a dramatic change in membrane surface adhesion force. Cytokine and chemokine production measured by multiplex ELISA demonstrated DEP-induced inflammatory responses in both cell types.

BRMS1 expression alters the ultrastructural, biomechanical and biochemical properties of MDA-MB-435 human breast carcinoma cells: an AFM and Raman microspectroscopy study.

Restoring BReast cancer Metastasis Suppressor 1 (BRMS1) expression suppresses metastasis in MDA-MB-435 human breast carcinoma cells at ectopic sites without affecting tumor formation at orthotopic site in the body. BRMS1 expression induces many phenotypic alterations in 435 cells such as cell adhesion, cytoskeleton rearrangement, and the down regulation of epidermal growth factor receptor (EGFR) expression. In order to better understand the role of cellular biomechanics in breast cancer metastasis, the qualitative and quantitative detection of cellular biomechanics and biochemical composition is urgently needed. In the present work, using atomic force microscopy (AFM) and fluorescent microscopy we revealed that BRMS1 expression in 435 cells induced reorganization of F-actin and caused alteration in cytoarchitectures (cell topography and ultrastructure). Results from AFM observed increase in biomechanical properties which include cell adhesion, cellular spring constant, and Young's modulus in 435/BRMS1 cells. Raman microspectroscopy showed weaker vibrational spectroscopic bands in 435/BRMS1 cells, implying decrease in concentration of cellular biochemical components in these cells. This was despite the similar spectral patterns observed between 435 and 435/BRMS1 cells. This work demonstrated the feasibility of applying AFM and Raman techniques for in situ measurements of the cellular biomechanics and biochemical components of breast carcinoma cells. It provides vital clues in understanding of the role of cellular biomechanics in cancer metastasis, and further the development of new techniques for early diagnosis of breast cancer.

Fluorescence resonance energy transfer-based method for detection of DNA binding activities of nuclear factor kappaB.

The DNA binding protein nuclear factor kappaB (NF-kappaB) and the cellular signaling pathways in which it participates are the central coordinators of many biological processes, including innate and adaptive immune responses, oxidative stress response, and aging. NF-kappaB also plays a key role in diseases, for example, cancer A simple, convenient, and high-throughput detection of NF-kappaB activation is therefore important for systematically studying signaling pathways and for screening therapeutic drug targets. We describe a method based on fluorescence resonance energy transfer (FRET) to directly measure the amount of activated NF-kappaB. More specifically, a double-stranded DNA (dsDNA) probe was designed to contain a pair of FRET fluorophores at the same end of the probe and an endonuclease binding site within the NF-kappaB consensus sequence. The activated NF-kappaB was detected by FRET following the restriction enzyme digestion. Using three different analyte materials--(i) purified recombinant NF- kappaB p50, (ii) nuclear extracts, and (iii) whole cell lysates--we demonstrated that this assay is as sensitive as the traditional, widely used electrophoretic mobility shift assay (EMSA), but much less labor-intensive for measuring NF-kappaB DNA binding activities. In addition, this FRET-based assay can be easily adapted for high-throughput screening of NF-kappaB activation.

Acoustic sensor for monitoring adhesion of Neuro-2A cells in real-time.

Neuronal adhesion plays a fundamental role in growth, migration, regeneration and plasticity of neurons. However, current methods for studying neuronal adhesion cannot monitor this phenomenon quantitatively in real-time. In this work, we demonstrate the use of an acoustic sensor to measure adhesion of neuro-blastoma cells (Neuro-2A) in real-time. An acoustic sensor consisting of a quartz crystal sandwiched between gold electrodes was placed in a flow cell and filled with 600 microl of phosphate buffered saline (PBS). Two sets of in vitro experiments were performed using sensors that had uncoated gold electrodes and sensors that were coated with a known neuronal adhesion promoter (poly-l-lysine or PLL). The instantaneous resonant frequency and the equivalent motional resistance of the acoustic sensor were monitored every second. Cell Tracker was used to confirm neuronal adhesion to the surface. Addition of 10 microl of media and Neuro-2A cells into the above set-up elicited exponential changes in the resonant frequency and motional resistance of the quartz crystal with time to reach steady state in the range of 2-11 h. The steady-state change in resonant frequency in response to addition of neurons was linearly related to the number of Neuro-2A cells added (R2=0.94). Acoustic sensors coated with the adhesion promoter, PLL showed a much higher change in resonant frequency for approximately the same number of neurons. We conclude that the acoustic sensor has sufficient sensitivity to monitor neuronal adhesion in real-time. This has potential applications in the study of mechanisms of neuron-substrate interactions and the effect of molecular modulators in the extra cellular matrix.