Role of locus coeruleus adrenergic neurons in breast cancer progression Kyle
1 Shutkind ,
1Donald
Adrian
2 Berisha ,
Jeremy Borniger,
2 PhD
and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Transsynaptic tracing with pseudorabies virus (PRV)
Background
3. ChR2 is localized to norepinephrine producing LC cells
ChR2-eYFP
• A growing body of evidence suggests that the sympathetic nervous system regulates breast cancer growth1,2 • The locus coeruleus (LC) is implicated in many neurological diseases, including depression, Alzheimer's, and Parkinson’s, but its potential role in cancer is unexplored • Goal of the current project: investigate whether aberrant activity of the LC contributes to enhanced tumor progression
TH
4V
eYFP
eYFP
Figure 3 | PRV was injected into the 9th mammary gland of mice to characterize neuronal connection between brain and mammary gland.
Results 1. Optogenetic activation of noradrenergic LC promotes arousal
TH
Overlay
4V
eYFP
Overlay
Result 3 | Representative images after IHC revealing expression of ChR2-EYFP (top) and control EYFP (bottom) in noradrenergic LC cells Figure 1 | LC efferent pathways and relevant functions3. The LC is in the pons of the brain stem and is the principal site for the brain’s synthesis of norepinephrine. The descending pathway (circled) contributes to sympathetic signaling within the peripheral nervous system.
4. Locus coeruleus contains PRV positive cells after injection into mammary gland
Hypothesis • •
Result 1 | Pupillometry was performed to assess for pupil dilation to validate successful viral injection and ChR2 expression.
Sustained stimulation of LC norepinephrine producing neurons will accelerate cancer progression Potential findings would support the claim that the brain and cancer in the periphery engage in bi-directional communication
2. Tumor growth throughout optogenetic stimulation
Result 4 | PRV virus expressing red fluorescent protein is visualized in LC 6 days after injection into mouse mammary gland
Experimental group
Methods
Conclusions •
Optogenetics timeline
Body weight change (%)
ChR2-eYFP and eYFP injections
Control group
•
EYFP 10
0 Figure 7: Independent CRISPR knockout of CDK4 or CDK6 does not cause dropout in most breast cancer cell lines -10 studied. -20 0
4
8 12 16 20 24 28 32 36 40 44 48
Days
Figure 2 |Experimental timeline. ChR2-eYFP is a fusion protein composed of ChR2 and eYFP used in experimental group. eYFP is used in control group. MMTV-PyMT cancer cells were injected to create a mouse model of human breast cancer.
Future Direction
ChR2-EYFP
20
Result 2 | Outcome of the six mice in the experiment. Mice body weight and tumor size was recorded each day during stimulation. Note one of the tumor injections failed in an experimental mouse and one control mouse died during the experiment.
Optogenetic stimulation of LC dilates pupil and IHC reveals successful expression of Cre-dependent ChR2-eYFP in the LC of TH-Cre mice PRV labeled neurons in the LC after mammary gland injection reveals potentially important connection between the LC and breast tissue
• • •
Repeat experiment in larger cohort of mice to generate statistical significance Quantify breast cancer metastasis to lungs and liver Perform fiber photometry of LC throughout breast cancer progression to assess breast cancer induced changes in the LC
Resources 1. Kamiya, A., Hayama, Y., Kato, S., Shimomura, A., Shimomura, T., Irie, K., ... & Ochiya, T. (2019). Genetic manipulation of autonomic nerve fiber innervation and activity and its effect on breast cancer progression. Nature neuroscience, 22(8), 1289-1305. 2. Sloan, E. K., Priceman, S. J., Cox, B. F., Yu, S., Pimentel, M. A., Tangkanangnukul, V., ... & Cole, S. W. (2010). The sympathetic nervous system induces a metastatic switch in primary breast cancer. Cancer research, 70(18), 7042-7052. 3. Bari, B. A., Chokshi, V., & Schmidt, K. (2020). Locus coeruleus-norepinephrine: basic functions and insights into Parkinson’s disease. Neural regeneration research, 15(6), 1006.