Lifu Xiao

Non-invasive detection of biomechanical and biochemical responses of human lung cells to short time chemotherapy exposure using AFM and confocal Raman spectroscopy

Cellular biomechanical properties including cell elasticity and cell adhesion are regarded as criteria to differentiate cancer cells and normal cells. In this study, the biomechanical properties including the Youngs modulus and adhesion force of human lung adenocarcinoma epithelial cell line A549 and non-cancerous human primary small airway epithelial cells (SAECs) were measured by using atomic force microscopy (AFM). It was found that primary SAECs are stiffer and more adhesive than cancerous A549 cells. Upon treatment with anti-cancer drug doxorubicin (DOX) for a short time (4 hours), both biomechanical properties of A549 cells were found to be increased while those of SAECs were decreased, implying that DOX-induced response mechanisms are different between these two types of cells (cancerous vs. primary cells). Using confocal Raman spectroscopy, we measured the changes in (sub)cellular biochemical compositions of both cell types before and after DOX exposure. Our ultimate goal is to find out the potential relationship between the changes in biomechanics and biochemical compositions of lung epithelial cells in response to anti-cancer drugs.

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.

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.

Label-free and non-invasive monitoring of porcine trophoblast derived cells: differentiation in serum and serum-free media

Traditional approaches to characterize stem cell differentiation are time-consuming, lengthy and invasive. Here, Raman microspectroscopy (RM) and atomic force microscopy (AFM) - both considered as non-invasive techniques - are applied to detect the biochemical and biophysical properties of trophoblast derived stem-like cells incubated up to 10 days under conditions designed to induce differentiation. Significant biochemical and biophysical differences between control cells and differentiated cells were observed. Quantitative real time PCR was also applied to analyze gene expression. The relationship between cell differentiation and associated cellular biochemical and biomechanical changes were discussed. Monitoring trophoblast cells differentiation.

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 Youngs 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.

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 Youngs 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.

AFM and Raman Spectroscopy, Applications in Cellular Imaging and Assays

By taking advantage of their respective technical capabilities, the combination of atomic force microscopy (AFM) and Raman spectroscopy offers new instrument tools that have the ability of conducting high-resolution imaging and chemical analysis of cellular samples. This combined approach opens a new horizon for cellular and molecular imaging at a single-cell level. In this article, some examples of Raman–AFM applications were illustrated, including embryonic stem cell differentiation monitoring, cellular composition changes of fatty acid-responsive cells, and cellular assay in microfluidic device.

Biochemical, biophysical, and genetic changes of porcine trophoblast-derived stem-like cells during differentiation as evaluated using Raman microspectroscopy, Atomic force microscopy, and quantitative polymerase chain reaction.

Porcine trophoblast‐derived stem‐like cells grown into serum medium start to differentiate and become senescent within 30 days. However, trophoblast‐derived cells, cultured in vitro in a defined and non‐serum medium, have the regenerative properties, such as indefinite passage and foreign DNA receptivity, similar to stem cells. To evaluate the biochemical, biophysical, and genetic changes of the terminal differentiation of trophoblast derived cells, Raman microspectroscopy, atomic force microscopy, and qPCR were applied. It was found that Raman spectral intensities of characteristic peaks, cell morphology, and Youngs modulus can be used to distinguish differentiated and undifferentiated trophoblast cells. In addition, 17 cytoskeleton and extracellular matrix‐related genes were significantly impacted by medium type (non‐serum versus serum). Our findings suggest that Raman microspectroscopy and atomic force microscopy—both considered as label‐free, non‐invasive techniques—can be applied to distinguish differentiated trophoblast cells, and cellular biochemical information and biophysical properties can be indicative of cellular differences during cell differentiation. In addition, most of cytoskeleton‐related genes exhibit similar pattern to that of Youngs modulus during trophoblast cell differentiation, indicating the potential connection between cytoskeleton‐related genes and cellular stiffness. genesis 53:749–761, 2015.