Metastatic brain tumors in breast cancer patients are incurable. A novel strategy to prevent central nervous system (CNS) metastasis is through targeting dormant disseminated tumor cells (DTCs), which seed the CNS as single cells early during tumor progression, evade immune targeting, lie dormant for years, and eventually give rise to lethal metastases. Immunotherapies designed to eliminate dormant DTCs before they form tumors could prevent incurable CNS recurrence, potentially prolonging survival of breast cancer patients. Preliminary work for this research proposal shows that phagocytosis of tumor cells by CNS-resident macrophages called microglia is inhibited by astrocytes, another type of glial cell in the CNS with important neuroprotective functions in health and development. We also used computational analyses to predict candidate astrocyte ligand::microglia receptor interactions, hypothesizing that these regulatory axes could explain how astrocytes inhibit microglial immunosurveillance within the CNS DTC niche. Using a lentiviral short hairpin RNA (shRNA) knockdown approach, we screened through a list of these receptors and identified promising candidates that negatively regulate microglial phagocytosis in culture. We will next test the functional effects of knocking out these receptors with respect to microglial phenotype and control of CNS metastatic burden in vivo. By addressing these topics, our research will define novel mechanisms of microglial regulation, and test whether therapeutic manipulation of these pathways restores CNS DTC immunosurveillance by microglia. These basic biological insights could then be leveraged to design innovative immunotherapies that eradicate DTCs and prevent lethal CNS metastasis in breast cancer patients.

In lung disease, cardiac dysfunction e.g. atrial arrhythmias is known to occur. Whether this is due to cross-talk in the stellate ganglion (SG), the source of efferent sympathetic drive to the heart and lungs, is unknown. The objective of this study is to determine if there is remodeling of heart- and lung-projecting SG neurons in pulmonary fibrosis (PF). Mice were given bleomycin or saline to provoke lung damage. While sedated, pulmonary and cardiac neuronal tracers were delivered. The stellates were then isolated, stained, and imaged. Our findings show that heart- and lung-projecting SG neurons colocalize in the craniomedial region. PF leads to increased size of heart- and lung-projecting SG neurons. The average size of heart-projecting neurons was 310μm^2 in saline (n=6) and 342μm^2 in bleomycin (n=6), p<0.0001. The average size of lung-projecting neurons was 274μm^2 in saline (n=8) and 305μm^2 in bleomycin (n=7), p=0.0141. PF leads to increased Neuropeptide Y (NPY) immunoreactivity for lung-projecting SG neurons and decreased NPY immunoreactivity in heart-projecting SG neurons. The fraction of total lung-projecting neurons immunoreactive for NPY was 59% in saline (n=158) and 79% in bleomycin (n=82), p=0.0012. The fraction of total heart-projecting neurons immunoreactive for NPY was 78% in saline (n=674) and 68% in bleomycin (n=731), p<0.0001. These findings demonstrate remodeling of cardiac sympathetic neurons in response to PF, and suggest the stellate ganglion is a site for heart-lung neural cross-talk.

Emerging research has shown that epidural electrical stimulation (EES) can be utilized to activate neural pathways involved in sensory and motor function in healthy individuals and those with spinal cord injuries. Although this seems to be a promising method of rehabilitation, the mechanism by which EES recruits neurons is unknown. In our previous research, we hypothesized that EES works by stimulating specific neuronal types and showed that somatostatin (SST) neurons are a type of neuron activated by EES. This raises the question of how neurons activated by EES are recruited. To address this, we performed immunohistofluorescence (IF) staining on tissues collected from mice that underwent EES. The mice had an endogenous fluorescent protein expressed by their SST neurons— Tandem dimer tomato (TdTomato)— along with the neuronal activation markers, cfos— which is labeled via IF staining— and DAPI— used to identify all living cells in the tissue. Previously, analysis of these signals were strictly qualitative, or they relied on manual counting. This experiment combined automated cell counting programs like Cell Profiler and ImageJ and our own formulated cell counting code with manual counting to optimize and streamline the quantification of living SST neurons that are activated by EES, as indicated by the colocalization of all three stains. Future applications of this research include determining the percentage of neurons activated by EES, quantifying the percentage of activated neurons that are SST, and investigating what other types of neurons can be recruited by EES and how they contribute to activating the neural circuits.

Epilepsy is a neurological disorder characterized by recurrent, unprovoked seizures. Pathogenic, gain-of-function mutations within SCN8A, a gene that encodes the ɑ-subunit of sodium channel Naᵥ1.6 causes neuronal hyperexcitation. This mutation is associated with an acute form of childhood epilepsy known as developmental epileptic encephalopathy-13 (DEE-13). Studies of epilepsy recurrently use rodent models; however, structural differences between the brains of rodents and humans may impact cellular and behavioral phenotypes. Brain organoid systems have been developed to replicate key features of the human brain’s neural networks and cellular architecture to mitigate some of these challenges. It is hypothesized that differences in cell-type expression patterns in the SCN8A-mutant organoids might lead to the pathology observed in DEE-13. To develop brain organoids, human-induced pluripotent stem cells (hiPSCs) must be adequately cultured with the proper growth factors, such as human fibroblast growth factor 2 (FGF-2), to ensure a robust quality of brain organoid production. This study completed a quality analysis of hiPSC mutant SCN8A cells and CRISPR/Cas9 corrected isogenic control hiPSCs treated with an FGF-2 DISC compared to untreated cells. The FGF-2 DISC releases heparin-binding growth factors over a weeklong period to promote pluripotency and prevent differentiation. Untreated cultures underwent hESC media changes by hand with daily FGF-2 replenishment. The health and proliferation of the DISC-treated hiPSCs were comparable to the untreated cells maintained by standard practices.