Label-Free Raman Imaging to Monitor Breast Tumor Signatures

Abstract

Although not yet ready for clinical application, methods based on Raman spectroscopy have shown significant potential in identifying, characterizing, and discriminating between noncancerous and cancerous specimens. Real-time and accurate medical diagnosis achievable through this vibrational optical method largely benefits from improvements in current technological and software capabilities. Not only is the acquisition of spectral information now possible in milliseconds and analysis of hundreds of thousands of data points achieved in minutes, but Raman spectroscopy also allows simultaneous detection and monitoring of several biological components. Besides demonstrating a significant Raman signature distinction between nontumorigenic (MCF-10A) and tumorigenic (MCF-7) breast epithelial cells, our study demonstrates that Raman can be used as a label-free method to evaluate epidermal growth factor activity in tumor cells. Comparative Raman profiles and images of specimens in the presence or absence of epidermal growth factor show important differences in regions attributed to lipid, protein, and nucleic acid vibrations. The occurrence, which is dependent on the presence of epidermal growth factor, of new Raman features associated with the appearance of phosphothreonine and phosphoserine residues reflects a signal transduction from the membrane to the nucleus, with concomitant modification of DNA/RNA structural characteristics. Parallel Western blotting analysis reveals an epidermal growth factor induction of phosphorylated Akt protein, corroborating the Raman results. The analysis presented in this work is an important step toward Raman-based evaluation of biological activity of epidermal growth factor receptors on the surfaces of breast cancer cells. With the ultimate future goal of clinically implementing Raman-guided techniques for the diagnosis of breast tumors (e.g., with regard to specific receptor activity), the current results just lay the foundation for further label-free optical tools to diagnose the disease.

Keywords: DNA denaturation; EGF overexpression; MCF-7 cells; RNA; Raman spectroscopy.

Figure 1.

Integrated Raman spectra of two confocal Raman mapping images: MCF-10A nontumorigenic control sample cell (black spectrum) and MCF-7 breast cancer control cell (red spectrum).

Figure 2.

Confocal Raman mapping images of untreated and EGF-treated MCF-7 cells for vibrational signatures of: (A, B) DNA/RNA, (C, D) cumulative distribution of 1240 and 1658 cm⁻¹ protein bands, (E, F) 2940 cm⁻¹ protein-associated feature, and (G, H) lipid band-centered around 2870 cm⁻¹. A bright yellow pseudocolor corresponds to a higher intensity. Also, the arrows in image (E) mark differences in content from image presented in (C), and comparison of (G) with (C) similarly indicate modifications associated with lipid content.

Figure 3.

A and B, Confocal Raman mapping images performed with Cluster Analysis software of MCF-7 cells from untreated and EGF-treated samples, respectively. C and D, Raman spectra associated with each cluster and only in vibrational regions of interest. The same color code is maintained for images and for spectra.

Figure 4.

Western blotting analysis of EGF stimulation of MCF-7 cells. The cells were starved for 24 hours prior to EGF exposure for 30 minutes and at a concentration of 100 ng/mL and then harvested with 3 different lysis buffers using BD Falcon cell scrapers. The #1 lysis buffer refers to RIPA-like, #2 to NP-40, and #3 to MILLIPLEX MAP for multiplexing. The cells under the control treatment were only treated with RPMI basal media. Amido black staining of the membrane (bottom) indicates even transfer of proteins, and the membrane was probed for phosphorylated p44 MAPK and phosphor-Akt. β-Actin was used as an additional loading control.