Biological Response to Microbeam Radiation Therapy

Stuart Hombsch , Ewan Blanch [1] [1],

Jeffry Crosbie , Oliver Jones , Jacqueline Donoghue , Leonie Cann
[1] [1] [2] [2]

[1]
RMIT University, University of Melbourne [2]

Background What is MRT?

External beam radiotherapy is one of the most versatile and common methods of cancer treatment. MRT is a preclinical radiotherapy technique that utilises synchrotron generated soft x-rays passed
By irradiating tumours with high energy photons, cancer cells can be killed effectively, but often the through a collimator to produce a characteristic ‘barcode’ pattern with areas of
dose limiting factor is tolerance of the surrounding healthy tissue to radiation. extremely high dose deposition called ‘peaks’ and areas of very low radiation exposure
called ‘valleys’. Peak areas are typically 4 μm wide, separated by valley regions about 400
Microbeam Radiation Therapy (MRT), a preclinical radiotherapy technique that employs spatially μm in width.
fractionated kilovoltage x-rays, has recently gained attention for its remarkable tissue sparing effects
while still achieving good tumour control. Understanding the radiation response mechanisms of Preclinical animal studies have shown many tissue types are able to tolerate peak doses
healthy tissue is an important step in maximising the efficacy of this important treatment. of MRT at least an order of magnitude higher than conventional radiotherapy[1]. The
underlying biological processes allowing these tissue sparing effects while still
This study aims to use Raman microscopy to examine the biochemical response of murine brain achieving good tumour control are poorly understood. Leading hypotheses include the
Figure 1: H&E stained tissue
tissue to both conventional broadbeam radiotherapy and MRT. participation of the immune system, preferential damage to tumour microvasculature of weaned piglet brain tissue
following MRT treatment
and cell signalling processes .
[1-3]
with a peak dose of 300 Gy. [4]

Methods Tissue Imaging

A total of 15 formalin fixed, paraffin embedded brain tissue sections from three treatment types Raman imaging experiments were used to look
(control, broadbeam irradiated (12 Gy) and microbeam irradiated (295 Gy peak dose)) were cut to 7 μ for different response mechanisms in peak and
m sections and placed on CaF2 slides for Raman analysis. valley regions of MRT treated tissues.

Raman spectra were acquired from the cerebellum of each tissue section in a raster scan pattern Clear track marks that can be seen in the
using a Horiba LabRAM HR Evolution Raman spectrometer, 50x dry objective lens (NA = 0.55), 600 optical image of the tissue due to peak MRT
l/mm diffraction grating with 30 second acquisition time per point and 550 – 1800 cm-1 spectral doses do not appear in the Raman image,
window. A 785 nm laser was used for excitation with an output of 100 mW. 400 μm indicating the biological response to MRT
extends far beyond the highly irradiated
Spectra were individually pre-processed in-house MATLAB algorithms before being analysed with Figure 2: Optical and Raman image of MRT treated murine brain tissue. Red, green regions.
and blue pixel intensity represent normalised magnitues of principal component
principal component analysis using standard MATLAB algorithms. scores 1, 2, and 3, respectivley.

Results
Mean Spectra
140
Control Raman Shift
* MRT (295 Gy peak)
Broadbeam MRT Band Assignment
120
Broadbeam (12 Gy)
(cm-1)
100 *
756 Decrease (weak) Decrease Tryptophan
Intensity

80

*
60 830 Increase O-P-O asym str/Tyr br
*
40 *
850 Decrease Increase Tyr ring br, glycogen
20

935 Decrease C-C sym str bk, α-helix

0
600 800 1000 1200 1400 1600
Wavenumber (cm-1) 1082 Decrease (weak) Increase Carbohydrate
Figure 3: Mean spectra of control, microbeam and conventionally irradiated tissues. Parrafin resudue is present in all samples even after de-waxing in xylene, denoted with *.

1207 Increase Increase Phenylalanine

1339 Decrease Decrease (weak) DNA

a b Loadings for PC 4
0.8
20
1665 Decrease Amide-I
Control
Principal Component 5 (1.6%)

Intensity

MRT
0.6 Broadbeam
10

0
0.4 Table 1: Notable differences in mean spectra of treatment types relative to the control spectrum. Band assignment was based on similar published
-10
studies[5-7].
0.2 -20
600 800 1000 1200 1400 1600

0 20
Loadings for PC 5
10
Intensity

-0.2

-0.4
-10
0

Conclusion
-20
-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 600 800 1000 1200 1400 1600
Principal Component 4 (2.1%) Wavenumber (cm ) -1
Group mean spectra reveal several subtle differences between each treatment type, many
Figure 4: a) PCA scatter plot showing grouping of all three treatment types. The contol contorl group the largest spread while there is a small amount of overlap between Broad-
beam and MRT treated tissues. b) Loadings plots of PC4 and 5 show minimal variation in AmideI-III bands, with most variation occuring below 1200 cm-1.
of which indicate changes in protein side chains, while differences in nucleic acids and
carbohydrates were also observed. Lipids of biological origin were not examined as
sources of variation as the paraffin removal process can artificially alter the lipid content of
tissues.

Acknowledgements These early results indicate that MRT induces a separate biochemical response to
conventional radiotherapy in healthy tissues. Future work includes repeating this
I would like to thank RMIT University for funding my PhD scholarship, and ICORS 2018 for awarding experiment on snap frozen tissues to examine the role of lipids in radiation response of
me a student travel grant to allow me to present my research. healthy tissues and additional Raman imaging experiments to confirm tissue response to
MRT is not confined to regions receiving the peak dose.

References
1. Smyth, L.M., et al., The normal tissue effects of microbeam radiotherapy: What do we know, and what do we need to know to plan a human clinical trial? Int J Radiat Biol, 2016. 92(6): p. 302-11. 2. Smith, R.W., et al., Proteomic changes in the rat brain induced by homogenous irradiation and by the bystander effect resulting from high energy synchrotron X-ray microbeams. Int J Radiat Biol,
2013. 89(2): p. 118-27. 3. Fontanella, A.N., et al., Effects of high-dose microbeam irradiation on tumor microvascular function and angiogenesis. Radiat Res, 2015. 183(2): p. 147-58. 4. Barber, H.B., et al., <title>Weanling piglet cerebellum: a surrogate for tolerance to MRT (microbeam radiation therapy) in pediatric neuro-oncology</title>. 2001. 4508: p. 65-73. 5. Harder, S.J., et al., Raman
spectroscopy identifies radiation response in human non-small cell lung cancer xenografts. Sci Rep, 2016. 6: p. 21006. 6. Meksiarun, P., et al., Breast cancer subtype specific biochemical responses to radiation. Analyst, 2018. 143(16): p. 3850-3858. 7. Matthews, Q., et al., Radiation-Induced Glycogen Accumulation Detected by Single Cell Raman Spectroscopy Is Associated with Radioresistance
that Can Be Reversed by Metformin. PLoS One, 2015. 10(8): p. e0135356.