Raman spectroscopy identifies radiation response in human non-small cell lung cancer xenografts

Abstract

External beam radiation therapy is a standard form of treatment for numerous cancers. Despite this, there are no approved methods to account for patient specific radiation sensitivity. In this report, Raman spectroscopy (RS) was used to identify radiation-induced biochemical changes in human non-small cell lung cancer xenografts. Chemometric analysis revealed unique radiation-related Raman signatures that were specific to nucleic acid, lipid, protein and carbohydrate spectral features. Among these changes was a dramatic shift in the accumulation of glycogen spectral bands for doses of 5 or 15 Gy when compared to unirradiated tumours. When spatial mapping was applied in this analysis there was considerable variability as we found substantial intra- and inter-tumour heterogeneity in the distribution of glycogen and other RS spectral features. Collectively, these data provide unique insight into the biochemical response of tumours, irradiated in vivo, and demonstrate the utility of RS for detecting distinct radiobiological responses in human tumour xenografts.

Figure 1. Radiation related Raman spectral changes identified using principal component analysis.

Principal components (PC) 1 (a) and 2 (c) from H460 tumour tissue and corresponding PC score box plots (Tukey style box plot, outliers left out for clarity, notches indicate 95% confidence interval for the median) for PC1 (b) and PC2 (d). A statistically significant shift to more negative median PC1 and PC2 score was found for irradiated tumours compared to unirradiated tumours. Dashed red line in (a) corresponds to the Raman spectrum of pure glycogen (inverted, sample obtained from Life Technologies Inc., Burlington, ON, Canada). Black boxes represent PC score for all spectra collected over four tumours in a single dose group (15 Gy; n = 1969, 5 Gy; n = 2279, 0 Gy; n = 2032). Grey boxes represent PC score for all spectra collected for an individual tumour. Statistical significance was tested using a two-sided Wilcoxon rank sum test to 5% significance level, ****p-value ≤ 0.0001, *p-value ≤ 0.05, n.s. not significant.

Figure 2. Comparison of principal components derived from Raman spectra of non-small cell lung cancer irradiated in vitro and in vivo.

Principal component (PC) 1 (a) and PC2 (b) obtained for the current ex vivo study (black line) overlayed with the corresponding component obtained in a previous in vitro study on the H460 cell line. In vitro data was derived from principal component analysis on a Raman data set of H460 cells cultured an irradiated in vitro to doses of 0, 2, 4, 6, 8, 10, 30 and 50 Gy as presented and described previously. Principal component 1 from this ex vivo study is highly correlated (Pearson’s r value = 0.95) to PC1 from the in vitro experiment, as is PC2 (Pearson’s r value = 0.85).

Figure 3. Spatial maps of principal component (PC) 1 score at three unique regions within a single tumour irradiated to 15 Gy.

The spatial variation of PC1 scores demonstrates tissue heterogeneity within an individual tumour. Map areas are 120 μm² for (a) and (b), 210 μm² for (c) and images are formed with 15 μm² pixels which correspond with the x and y step sizes during Raman map acquisition. Pixels with slashes indicate pixels for which no PC score information is available because the spectrum at that pixel was excluded from PCA (for reasons discussed in methods section).

Figure 4. Periodic acid–Schiff (PAS) staining images from irradiated and unirradiated tumour tissue and liver tissue control.

A qualitative indication of increased glycogen content in irradiated (15 Gy) tissue compared to unirradiated (0 Gy) tissue was given by PAS staining. Images are collected at 20× magnification. PAS stained (a) Liver tissue (positive control) without diastase (alpha-amylase) and (d) with diastase. (b) Unirradiated control (0 Gy) H460 tumour tissue without diastase (alpha-amylase) and (e) with diastase. (c) Irradiated (15 Gy) H460 tumour tissue without diastase (alpha-amylase) and (f) with diastase.