Cell Phase Identification in a Three-Dimensional Engineered Tumor Model by Infrared Spectroscopic Imaging

Abstract

Cell cycle progression plays a vital role in regulating proliferation, metabolism, and apoptosis. Three-dimensional (3D) cell cultures have emerged as an important class of in vitro disease models, and incorporating the variation occurring from cell cycle progression in these systems is critical. Here, we report the use of Fourier transform infrared (FT-IR) spectroscopic imaging to identify subtle biochemical changes within cells, indicative of the G1/S and G2/M phases of the cell cycle. Following previous studies, we first synchronized samples from two-dimensional (2D) cell cultures, confirmed their states by flow cytometry and DNA quantification, and recorded spectra. We determined two critical wavenumbers (1059 and 1219 cm^-1) as spectral indicators of the cell cycle for a set of isogenic breast cancer cell lines (MCF10AT series). These two simple spectral markers were then applied to distinguish cell cycle stages in a 3D cell culture model using four cell lines that represent the main stages of cancer progression from normal cells to metastatic disease. Temporal dependence of spectral biomarkers during acini maturation validated the hypothesis that the cells are more proliferative in the early stages of acini development; later stages of the culture showed stability in the overall composition but unique spatial differences in cells in the two phases. Altogether, this study presents a computational and quantitative approach for cell phase analysis in tissue-like 3D structures without any biomarker staining and provides a means to characterize the impact of the cell cycle on 3D biological systems and disease diagnostic studies using IR imaging.

Figure 1.

Experimental workflow, including synchronized 2D and 3D MCF10A cell cultures, sample preparation, and FT-IR spectral imaging. The subsequent data analyses are also shown in the flow chart.

Figure 2.

Validation of cell synchronization and IR spectral signatures associated with intracellular DNA levels. (A) Flow cytometry assessment of intracellular DNA content of cell samples. (B) Quantification of extracted DNA from different cell phases. (C) Average IR spectra of cells recorded from asynchronous (blue), G1/S arrested (green), and G2/M arrested (purple) phases. Standard deviation is marked as the shaded area. (D) Absorbance visualization with wavenumber as categorical labels to identify the most significant spectral bands that distinguish between cell phases, with absorbance at (E) 1059 cm⁻¹ (left) and 1219 cm⁻¹ (right) showing significant differences but considerable overlap as well. For the Box–Whisker plots in panels (D) and (E), the top and bottom edges correspond to the first and third quartiles and the midline represents the sample median. The upper and lower whiskers extend from the hinge up to 1.5× IQR. Outlier values are indicated if they extend beyond this range.

Figure 3.

(A) Experimental setup for the MCF10A 3D culture. (B) Systematic figure of MCF10A acini formation under 3D cell-embedded conditions. (C) Size variation of M1 to M4 acini throughout 15 days of culture. (D) (Top row) Bright-field image of MCF10A acini formation at days 5, 10, and 15 of culture. Scale bar (white): 200 μm; inset diagrams show zoomed-in images of the acinus. Scale bar (black): 100 μm. (Bottom row) H&E-stained images of cryosectioned acini fixed at days 5, 10, and 15 of culture. Dark purple and pink stains indicate the nucleus and cytoplasm, respectively. Scale bar: 200 μm.

Figure 4.

Label-free identification of cell phases in a single acinus. (A) Chemical images of cryosectioned acini shown for the ratio of absorbance at 1219–1640 cm⁻¹ and acquired at days 5, 10, and 15 during the 3D cell culture duration. Scale bar: 50 μm. Corresponding intensity histograms are shown with the median and interquartile range above. The medians are 0.7559, 0.6794, and 0.6416, respectively, on days 5, 10, and 15. (B, C) Virtual staining of MCF10A and M2 acinus based on K-means clustering. Green, purple, and gray colors refer to G1/S, G2/M, and outliers, respectively. Scale bar: 100 μm. (D) Difference in absorbance spectra for days 5 and 10 and days 5 and 15. The blue line shows the difference between days 5 and 10, while the orange line shows the difference between days 5 and 15. The shaded areas present the standard deviation. (E) Analysis of the cell population in G1/S and G2/M phases for single acini at days 5, 10, and 15 for MCF10A cells (solid color) and M2 cells (solid color with white stripes). For MCF10A, the fraction of the cells in the G1/S phase changed from 51.9 to 66.73% during 15 days of culture. On the other hand, for M2 cells, the fraction of the cells in the G1/S phase has a more drastic shift from 6.34 to 90.24%.