Why Microbeams? - raraf

Why Microbeams
A microbeam can deposit ionizing radiation damage
in microscopic or sub-microscopic regions of cells
David J Brenner
Center for Radiological Research
Columbia University Medical Center
djb3@columbia.edu

Why Microbeams
A microbeam can deposit ionizing radiation damage
in microscopic or sub-microscopic regions of cells
Allows investigation of single-particle effects
Allows investigation of intra- and inter-cellular
mechanisms of stress response

Single-particle effects: Radon

Single-particle effects: Radon
1518 Woodcut:
Doctor and
nurse treating a
sick miner at
the Joachimsthal
hospital

Radon-Related Rationale for Microbeam:
Number of target cells exposed to a
given number of alpha particle traversals
# of
traversals
Colorado
miners
Domestic
radon
exposure
“Conventional”
in vitro
experiments
0 700,000 100,000,000 37,000
1 3,500,000 20,000 37,000
2 8,500,000 0 18,000
3 14,000,000 0 6,000
4 17,000,000 0 1,500
5 17,000,000 0 300

Oncogenic transformation by exactly one alpha
particle and a Poisson mean of one alpha particle
Transformation Frequency
(Miller et al 99)

Why Microbeams
A microbeam can deposit ionizing radiation damage
in microscopic or sub-microscopic regions of cells
Allows investigation of single-particle effects
Allows investigation of intra- and inter-cellular
mechanisms of stress response

Cytoplasmic irradiation

Why Microbeams
A microbeam can deposit ionizing radiation damage
in microscopic or sub-microscopic regions of cells
Allows investigation of single-particle effects
Allows investigation of intra- and inter-cellular
mechanisms of stress response

“Providing researchers with tools and
capabilities to make new discoveries”
Single-cell microbeams
Intra-cellular studies
Inter-cellular studies
Single cells Cells Tissues Animals

A quantitative example of inter-cellular damage communication:
Bystander Responses
Damage is expressed in “bystander” cells,
which are near to an irradiated cell, but have not
themselves received any energy deposition

Bystander effects have been reported for a
variety of endpoints using single-cell systems
Sister-chromatid exchanges
Cell killing (mitotic and apoptotic)
Micronucleus induction
Mutation induction
In-vitro
oncogenic transformation
Changes in gene expression
Altered cell growth
And for almost every radiogenic endpoint
that has been studied!

Why is there so much interest in
bystander effects
1. Basic interest in damage signal transduction
2. Practical interest in low-dose risk estimation..

Low-dose risk estimation
and the bystander effect
Induced CD59 - Mutants per 10 5 Survivors
140
120
100
80
60
40
20
Measured mutations
0
0 20 40 60 80 100
Percent of Cells Irradiated with One Alpha Particle
Based on mutation data from the
RARAF microbeam.
Zhou et al PNAS 98, 14410-5 (2001)
• Where bystander responses
have been quantitated,
they have shown saturation
• In such cases,
extrapolating linearly from
low to very low doses
could underestimate the
risk at very low doses.

Various experimental approaches to
bystander studies
Irradiate with a broad beam of high-LET
radiation at a very low dose, such that most
cells not hit
Intra-media signal transfer
‣ Irradiate cells/medium, then transfer irradiated
medium/cells onto fresh cells
‣ Co-culturing dishes
Microbeam studies:
‣ Hit only specified cells in the field

Early Bystander Studies
at the Columbia Microbeam
Shoot α particles at the
fibroblasts with blue-stained
nuclei, but not at those with
red-stained cytoplasm,
then score micronuclei
Frequency of micronuclei:
• Controls 0.8±0.6%
• Hit cells 30±4%
• Non-hit cells 5±1%

We can hit a predetermined fraction of cells...

In-vitro oncogenic transformation with microbeam
White: All cells hit by α particles
Yellow: Only 1 in 10 cells hit
Observed frequency / 10 4 surviving cells
16
14
12
10
8
6
4
2
0
Semi-confluent
single-cell monolayer
0 1 2 3 4 5 6 7 8
# of α particles through hit cells
Miller et al.
PNAS

We can kill a defined fraction of cells on a dish,
the remainder not being hit….
20 α-particles
X
X
20 α-particles through the nucleus kills the cells that are hit (SF< 1%)
Mutations observed therefore come from unhit “bystander” cells.

Mutations in A L cells:
20% of the cells each hit by 20 α particles
Mutation Frequency
T. K. Hei PNAS

Most bystander studies
have been performed with
single-cell systems
In that bystander effects involve
cell-to-cell communication,
it is important to study these effects
in normal three-dimensional human
tissue

Microbeam-based bystander studies in
human artificial 3-D skin

Microbeam-based bystander experiments
in human 3-D tissue systems

Apoptosis
Micronuclei
Fraction of apoptotic cells
0.06
0.04
0.02
Bystanders
Controls
0.00
100 300 500 700 900 1100
Distance from irradiated cells (microns)
Fraction of cells with micronuclei
0.03
0.02
Unirradiated bystanders
Controls
Bystanders
Controls
0.01
100 300 500 700 900 1100
Distance from irradiated cells (microns)
Belyakov et al PNAS 102, 14203-8 (2005)
For both apoptosis and micronucleus induction,
the range of the bystander effect in tissue is about
1 mm, or 50 to 100 cells
The average enhancement in effect, over this
range of distances, is about 1.6 for micronuclei
and 2.8 for apoptosis.

α-particle microbeam irradiation – 1 hour post-irradiation
Phosphorylation of p53 at Ser 15
0.35
0.3
Zone of Irradiation
Intensity (AU)
0.25
0.2
0.15
0.1
• Individual bystander / irradiated
cells in partially irradiated tissue
O Individual cells in control tissue
Individual Cells in Control Tissue
Individual Cells in Irradiated Tissue
0.05
0
-10000 -500 10000 1500 1000 2000
Distance from Distance irradiated (mm) cells (mm)

The mechanisms underlying
bystander effects
Due to unexpected circumstances
this key slide cannot yet be shown

The growth of single-cell microbeams
1
Gray Laboratory, London
2
3
4
Dublin, Ireland**
5
Stresa, Italy
6
Oxford, England
7
International Workshops on Microbeam
Probes of Cellular Radiation Response
Pacific Northwest Labs, Washington
Columbia University, New York**
Columbia University, New York**
Number of
Year
microbeams
1993
3
1995
3
1997
4
1999
7
2001
12
2003
17
2006
28
8
Chiba, Japan
2008
32
10
7
9 Darmstadt, Germany 2010 36
Number of single-cell microbeams
30
20
10 Columbia University, New York** 2012
40
0
1993 1995 1997 1999 2001 2003 2005 2007 2009

SINGLE-CELL MICROBEAMS, 2006
LABORATORY LET ON LINE BIOLOGY
Columbia University I, New York high Yes Yes
Columbia University II, New York low to very high Yes Yes
Gray Laboratory, London low, high Yes Yes
Gray Laboratory, London soft X Yes Yes
JAERI, Takasaki, Japan high Yes Yes
PNL, Richland, Washington low Yes Yes
GSI, Darmstadt, Germany high Yes Yes
Texas A&M, College Station low Yes No
PTB, Braunschweig, Germany low, high Yes Yes
Bordeaux, France low, high Yes No
Padua, Italy low, high Yes No
Leipzig, Germany low, high Yes No
MIT, Boston low, high Yes No
Munich, Germany low, high Yes No
L’Aquila, Italy soft X No No
Padua, Italy high Yes No
LBL, Berkeley very high No No
University of Maryland low Yes Belfast, No N. Ireland
Lund, Sweden low, high Yes Chiba, No Japan
Tsukuba, Japan soft X Yes No
Tsuruga, Japan
Nagatani, Japan Low, high Yes Yes
Seoul, South Korea Low Yes McMaster, Yes Canada
..Helsinki, Finland High No Nagasaki, No Japan
Chapel Hill, North Carolina Low No No
Hefei, China
Saclay, France Low, high Yes No
Cracow, Poland Low Yes Shanghai, Yes China
Gradignan, France High Yes Yes
Lanzhou, China
Surrey University, UK Low, High No No

The Single-Cell Microbeam at the Radiological
Research Accelerator Facility - RARAF
A biologist-friendly facility for probing
intra-cellular and inter-cellular communication