1. Object recognition impaired in treated mice and mice with tumors
A novel preclinical model of cognitive and neuroinflammatory consequences of radiation and immunotherapy
GJ McGinnis 1,2,3, D Friedman 5, KH Young 5, CR Thomas, Jr. 3, M Gough 3,5, J Raber 2,3,4
1 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
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.
Cortex levels of pro-inflammatory cytokines change with treatment in mice without tumors
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.
Object recognition impaired in treated mice and mice with tumors
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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).
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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.
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.
Neuroinflammation is seen in mice treated with immunotherapy alone and radiation combined with immunotherapy
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Copyright Colin Purrington (
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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 20-plex (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).
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
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.
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