Transcript Cognitive and neuroinflammatory consequences of radiation and immunotherapy

GJ McGinnis
1
1,2,3,
D Friedman 5, KH Young 5, CR Thomas, Jr. 3, M Gough
3,5,
J Raber
2,3,4
Howard Hughes Medical Institute, 2Department of Behavioral Neuroscience, 3 Department of Radiation Medicine, Knight Cancer Institute 4 Department of Neurology and Division of Neuroscience,
Oregon National Primate Research Center, Oregon Health & Science University
3 Earle A. Chiles Research Institute, Providence Cancer Center, Portland, Oregon, USA
Introduction
Results
Up to one-third of cancer patients report cancer-related cognitive and
behavioral changes in the months or years following treatment1. These changes
can develop in patients receiving radiation treatment alone2.
Neuroinflammation has been identified as a mediator of cognitive and
behavioral dysfunction3.
Novel combinations of immunotherapy and radiation therapy have excellent
efficacy with regard to tumor outcomes (Figure 1), however a gap exists as to
potential effects on the brain.
(a)
No treatment
RT
αCTLA4
αCTLA4 +RT
Object recognition impaired in treated mice
and mice with tumors
Day 2
Day 1
(b)
Day 2
Exploration
20Gy
Leg diameter
Cortex levels of pro-inflammatory
cytokines change with treatment in mice
without tumors
15Gy
10Gy
5Gy
2.5Gy
0.02Gy
50
100
50
100
50
100
50
100
Time (days)
Figure 1. (a) CT-guided treatment plans using Small Animal Radiation
Research Platform (SARRP). (b) Tumor outcomes following treatment with
radiation (RT) and/or immunotherapy (αCTLA4).
Materials and methods
Lung (3LL) tumors were established subcutaneously in 2-4mo female
C57BL/6 mice. Mice then received either sham, radiation, immunotherapy, or
combined treatment (Figure 2). 10 mice per group.
Tumor
-
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-
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+
+
+
+
Tumor - - - - + + + +
- - - - + + + +
- - - - + + + +
RT
-
+
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+
-
+
-
+
RT - + - + - + - +
- + - + - + - +
- + - + - + - +
αCTLA4
-
-
+
+
-
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+
+
αCTLA4 - - + + - - + +
- - + + - - + +
- - + + - - + +
Figure 3. Novel Object. Mice were given access to two identical objects and 24 hours
later reintroduced to one familiar object (orange hex) and one novel object (green
triangle) (n = 10). In mice without tumors, object recognition was seen in every group
except mice receiving both treatments. In mice with tumors, object recognition was
only observed in mice treated with RT alone.
Figure 4. Cytokine Multiplex Immunoassay. Freshly dissected cortex
samples were homogenized and used for immunoassay (n = 4). Increased
levels of IFN-γ, IL-2, and FGF-Basic were seen in mice without tumors
receiving anti-CTLA4 treatment.
Neuroinflammation is seen in mice treated with immunotherapy alone and radiation
combined with immunotherapy
(a)
(b)
Dentate Gyrus
RT
αCTLA4
Tumor
No
tumor
NT
αCTLA4+
RT
Immunotherapy consisted of checkpoint inhibition using an anti-CTLA-4
antibody (αCTLA4; Clone #9D9, BioXCell). 20Gy focal radiation was
delivered using the SARRP (XStrahl). Dose was delivered using a
10mmx10mm collimator at a 50°angle (Figure 1).
Behavioral and cognitive testing was performed on all mice following
treatment. Tests included Rotarod, Open Field, Novel Object, Water Maze, and
Fear conditioning.
Six mice per group were perfused with 4% PFA for immunohistochemistry.
Brains were removed, transferred to 4% paraformaldehyde (PFA) overnight,
and stored in 30% sucrose. Fixed brains were coronally sectioned at 40μm into
five series using a cryostat. Sections were processed for CD-68 (Abcam
ab53444, 1:500) immunoreactivity to identify reactive microglia.
Fresh brain tissue was taken for four mice per group. Samples were
homogenized in PBS and used in an inflammatory cytokine and chemokine 20plex (GM-CSF, TNF-α, IL-2, IL-1β, IL-4, MIG, KC, VEGF, IL-17, MIP-1α,
IL-12, IL-10, IL-6, IL-5, FGF-Basic, IL-1α, IFN-γ, IL-13, MCP-1, and IP-10).
Tumor - - - - + + + +
- - - - + + + +
- - - - + + + +
- - - - + + + +
RT - + - + - + - +
- + - + - + - +
- + - + - + - +
- + - + - + - +
αCTLA4 - - + + - - + +
- - + + - - + +
- - + + - - + +
- - + + - - + +
Conclusions
In mice without tumors, object
recognition is lost following
treatment with combined
radiotherapy and immunotherapy.
This indicates the importance of
including healthy mice (without
tumors) in assessing the effects of
novel combination therapies on
the brain. These results may have
clinical significance in treatment
planning.
Loss of object recognition in
all mice with tumors except those
treated with radiotherapy
demonstrates a potential baseline
difference in behavior of mice
with tumors. This reflects clinical
data that show cognitive and
behavioral changes may
accompany cancer alone 1.
Changes in pro-inflammatory
cytokines in the brain following
treatment support the idea that
cognitive impairment may be
immune-mediated.
Both with and without tumors,
mice treated with immunotherapy
alone or immunotherapy and
radiotherapy demonstrate
increased microglial activation.
Increased microglial activation is
concerning; especially when
chronic, microglial activation has
been associated with
neurodegenerative conditions 5.
Figure 5. CD-68 immunoreactivity. (a) Representative images from the dentate gyrus. (b) Mean with SEM percent of total area occupied by CD-68 immuno-positive cells
in the dentate gyrus (hippocampus), cortex, CA1 (Cornu Ammonis) (hippocampus), and CA3 (Cornu Ammonis 3) (hippocampus) (n=6).
Acknowledgments
Literature cited
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Phillips, KM et al. (2012). Cognitive functioning after cancer treatment: a 3-year
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de Moor, JS et al. (2013). Cancer survivors in the United States: prevalence across the
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This project was made possible by support
from an HHMI Medical Research
Fellowship, a Collins Medical Trust, an
RSNA Research Medical Student Grant, an
N.L. Tartar Research Fellowship, an
OSLER TL1 Trainee grant, the William
Moss Kenneth Stevens Academic
Development Fund of the Department of
Radiation Medicine, and the development
account of Dr. Raber.