Three-dimensional (3D) tumor cell cultures grown in laminin-rich-extracellular matrix (lrECM) are considered to reflect human tumors more realistic as compared to cells grown as monolayer on plastic. Here, we systematically investigated the impact of ECM on phenotype, gene expression, EGFR signaling pathway, and on EGFR inhibition in commonly used colorectal cancer (CRC) cell lines. LrECM on-top (3D) culture assays were performed with the CRC cell lines SW-480, HT-29, DLD-1, LOVO, CACO-2, COLO-205 and COLO-206F. Morphology of lrECM cultivated CRC cell lines was determined by phase contrast and confocal laser scanning fluorescence microscopy. Proliferation of cells was examined by MTT assay, invasive capacity of the cell lines was assayed using Matrigel-coated Boyden chambers, and migratory activity was determined employing the Fence assay. Differential gene expression was analyzed at the transcriptional level by the Agilent array platform. EGFR was inhibited by using the specific small molecule inhibitor AG1478. A specific spheroid growth pattern was observed for all investigated CRC cell lines. DLD-1, HT-29 and SW-480 and CACO-2 exhibited a clear solid tumor cell formation, while LOVO, COLO-205 and COLO-206F were characterized by forming grape-like structures. Although the occurrence of a spheroid morphology did not correlate with an altered migratory, invasive, or proliferative capacity of CRC cell lines, gene expression was clearly altered in cells grown on lrECM as compared to 2D cultures. Interestingly, in KRAS wild-type cell lines, inhibition of EGFR was less effective in lrECM (3D) cultures as compared to 2D cell cultures. Thus, comparing both 2D and 3D cell culture models, our data support the influence of the ECM on cancer growth. Compared to conventional 2D cell culture, the lrECM (3D) cell culture model offers the opportunity to investigate permanent CRC cell lines under more physiological conditions, i.e. in the context of molecular therapeutic targets and their pharmacological inhibition.

Label-free chemical characterization of single cells is an important aim for biomedical research. Standard Raman spectroscopy provides intrinsic biochemical markers for noninvasive analysis of biological samples but is often hindered by the presence of fluorescence background. In this paper, we present an innovative modulated Raman spectroscopy technique to filter out the Raman spectra from the fluorescence background. The method is based on the principle that the fluorescence background does not change whereas the Raman scattering is shifted by the periodical modulation of the laser wavelength. Exploiting this physical property and importantly the multichannel lock-in detection of the Raman signal, the modulation technique fulfills the requirements of an effective fluorescence subtraction method. Indeed, once the synchronization and calibration procedure is performed, minimal user intervention is required, making the method online and less time-consuming than the other fluorescent suppression methods. We analyze the modulated Raman signal and shifted excitation Raman difference spectroscopy (SERDS) signal of 2 mum-sized polystyrene beads suspended in a solution of fluorescent dye as a function of modulation rate. We show that the signal-to-noise ratio of the modulated Raman spectra at the highest modulation rate is 3 times higher than the SERDS one. To finally evaluate the real benefits of the modulated Raman spectroscopy, we apply our technique to Chinese hamster ovary cells (CHO). Specifically, by analyzing separate spectra from the membrane, cytoplasm, and nucleus of CHO cells, we demonstrate the ability of this method to obtain localized sensitive chemical information from cells, away from the interfering fluorescence background. In particular, statistical analysis of the Raman data and classification using PCA (principal component analysis) indicate that our method allows us to distinguish between different cell locations with higher sensitivity and specificity, avoiding potential misinterpretation of the data obtained using standard background procedures.

In this work, the effects of thalassemia, a blood disease quite diffuse in the Mediterranean sea region, have been investigated at single cell level using a Raman Tweezers system. By resonant excitation of hemoglobin Raman bands, we have examined the oxygenation capability of beta-thalassemic erythrocytes. A reduction of this fundamental erythrocyte function has been found. The measurements have been performed on a significant number of red blood cells; the relative statistical analysis is presented. Moreover, the response to photo-induced oxidative stress of diseased cells with respect to the normal ones has been analyzed. Finally, the deformability of thalassemic erythrocytes has been quantified by measuring the membrane shear modulus by using a double-trap system: the measurements have revealed an increase in membrane rigidity of more than 40%, giving evidence that the genetic defect associated to thalassemia, which manly relies on hemoglobin structure, also strongly affects the erythrocyte mechanical properties. Our results demonstrate that the developed set-up may have potential for the monitoring of blood diseases and their response to drug therapies.

Raman spectroscopy permits probing of the molecular and chemical properties of the analyzed sample. However, its applicability has been seriously limited to specific applications by the presence of a strong fluorescence background. In our recent paper [Anal. Chem. 82, 738 (2010)], we reported a new modulation method for separating Raman scattering from fluorescence. By continuously changing the excitation wavelength, we demonstrated that it is possible to continuously shift the Raman peaks while the fluorescence background remains essentially constant. In this way, our method allows separation of the modulated Raman peaks from the static fluorescence background with important advantages when compared to previous work using only two [Appl. Spectrosc. 46, 707 (1992)] or a few shifted excitation wavelengths [Opt. Express 16, 10975 (2008)]. The purpose of the present work is to demonstrate a significant improvement of the efficacy of the modulated method by using different processing algorithms. The merits of each algorithm (Standard Deviation analysis, Fourier Filtering, Least-Squares fitting and Principal Component Analysis) are discussed and the dependence of the modulated Raman signal on several parameters, such as the amplitude and the modulation rate of the Raman excitation wavelength, is analyzed. The results of both simulation and experimental data demonstrate that Principal Component Analysis is the best processing algorithm. It improves the signal-to-noise ratio in the treated Raman spectra, reducing required acquisition times. Additionally, this approach does not require any synchronization procedure, reduces user intervention and renders it suitable for real-time applications.