Label-free discrimination of tumorigenesis stages using in vitro prostate cancer bone metastasis model by Raman imaging

Abstract

Metastatic prostate cancer colonizes the bone to pave the way for bone metastasis, leading to skeletal complications associated with poor prognosis and morbidity. This study demonstrates the feasibility of Raman imaging to differentiate between cancer cells at different stages of tumorigenesis using a nanoclay-based three-dimensional (3D) bone mimetic in vitro model that mimics prostate cancer bone metastasis. A comprehensive study comparing the classification of as received prostate cancer cells in a two-dimensional (2D) model and cancer cells in a 3D bone mimetic environment was performed over various time intervals using principal component analysis (PCA). Our results showed distinctive spectral differences in Raman imaging between prostate cancer cells and the cells cultured in 3D bone mimetic scaffolds, particularly at 1002, 1261, 1444, and 1654 cm^-1, which primarily contain proteins and lipids signals. Raman maps capture sub-cellular responses with the progression of tumor cells into metastasis. Raman feature extraction via cluster analysis allows for the identification of specific cellular constituents in the images. For the first time, this work demonstrates a promising potential of Raman imaging, PCA, and cluster analysis to discriminate between cancer cells at different stages of metastatic tumorigenesis.

Figure 1

Raman spectra of prostate cancer cells. (A) Averages of 20 spectra from 4 samples (2D PCa and 3D MSCs + PCa SCs d(X + Y), where X = 23 days of MSCs culture and Y = 5, 10, and 15 days of cancer cell culture after 23 days of MSCs culture) are shown in bold and overlaid on representative examples of spectra for each sample. Spectra are color-grouped according to culture type and duration of culture. (B) The corresponding difference spectra were calculated from the averaged Raman spectra between 2D culture and three different 3D cultures.

Figure 2

PCA analysis of Raman spectra. (A) PCA score plot spanned by PC1 and PC2, illustrating the intrinsic clustering of 2D PCa and 3D MSCs + PCa SCs. (B) The first two PCs accounting for 92.9% of the total variation in the Raman spectral dataset, revealing the significant Raman spectral features for the classification of cancer cells.

Figure 3

Calculation of Raman band intensity. Twenty cell spectra from each sample were selected for intensity calculation of chosen Raman bands. All spectra were normalized to DNA band at 780 cm⁻¹ before analysis. Histogram displaying intensities of Raman bands (A) 1002 cm⁻¹, (B) 1261 cm⁻¹, (C) 1444 cm⁻¹, and (D) 1654 cm⁻¹ across samples. (n = 20, one-way ANOVA followed by post hoc Tukey test, *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant difference between 2D PCa and 3D MSCs + PCa SCs; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 indicate significant difference between day (23 + 5), and other days of 3D MSCs + PCa SCs; ^$p < 0.05, ^$$p < 0.01, and ^$$$p < 0.001 indicate significant difference between day (23 + 10) 3D MSCs + PCa SC and day (23 + 15) 3D MSCs + PCa SC).

Figure 4

Raman maps of prostate cancer cells. Raman mapping images obtained from 2D PCa and three 3D MSCs + PCa SCs for vibrational signatures of: (A) 1002 cm⁻¹, (B) 1261 cm⁻¹, (C) 1444 cm⁻¹, and (D) 1654 cm⁻¹. Scale bars, 10 µm (A–D).

Figure 5

Cluster analysis of 2D PCa and PCa cells seeded on 3D bone-mimetic scaffolds for 5, 10, and 15 days. (A) optical images of samples used in Raman Imaging, (B) clusters indicating actin-rich regions colored in red, and the red stripe on the top of the image constitutes digitally introduced reference actin. (C) clusters indicating DNA rich regions colored in blue and the blue stripe on the top of the image constitutes digitally introduced reference DNA. Scale bars, 10 µm (A–C). (D) average spectra from ten randomly collected spectra from the actin-rich regions on day 15 and the reference spectra for actin, (E) average spectra from ten randomly collected spectra from the DNA rich regions on day 15 and the reference spectra for actin. The bone-mimetic scaffolds are created by seeding MSCs on 3D nanoclay based scaffolds and culturing for 23 days for the regeneration of bone tissue. PCa cells are seeded after the MSC culture period of 23 days is completed. The 2D PCa cells indicate PCa cells not seeded on scaffolds.