Renal Cell Carcinoma (RCC) kills more than 14,800 people annually in the United States alone. While immune and targeted therapies have shown some promise, late stage RCC is an aggressive and lethal disease, and so, new therapies that can treat RCC are urgently needed. TRAIL (Tumor necrosis factor (TNF)-related apoptosis-inducing ligand) has been shown previously to induce apoptosis in cancer cells by binding to TRAIL receptors DR4 and DR5. Unfortunately, DR5 antibodies have failed to improve patient survival in phase II clinical trials due, in part, to the inability to cluster enough membrane receptors and kill RCC cells in the body. This project is investigating the effects that computationally designed protein nanocages bound to DR5 antibodies have on kidney cancer cells in culture. In collaboration with the Institute for Protein Design, we have recently shown that these nanoparticles induce significant activation of caspase 8 and caspase 3 in renal carcinoma TRAIL resistant cells, but not in healthy primary kidney tubular cells. To further investigate the nanocage-induced apoptosis specificity to DR5 and the involvement of other TRAIL receptors, we used CRISPR/Cas9 to generate knockouts of TRAIL receptors in RCC. We are also investigating the effects of these nanocages on other downstream pathways including the NFkB and cFLIP pathways. This project advances the knowledge of mechanisms involved in TRAIL receptor-mediated apoptosis, and our results suggest a potential new treatment for RCC.

The ability to respond to and recover from a physically stressful event is defined as physical resilience. Because of the inherent individual variation in response to a specific stressor with increasing age, the ability to document resilience at a younger age would likely predict that an individual would be more resilient at an older age. In a preliminary experiment, this concept was tested in a mouse model of wound healing consisting of a through and through 2 mm biopsy within the central area of each ear. A cohort of 20 female and 20 male C57BL/6 mice (an inbred mouse strain used extensively in aging research), 18 months of age, were biopsied and the area of closure measured after two weeks using a digital imaging procedure. The area of closure showed that the biopsy opening healed at variable rates depending on each individual mouse independent of sex, but clustering resulted in two main groups of approximately equal numbers, a fast-healing group and a slow-healing group. The data were analyzed by me (student first author), and I was able to assess the correlation of the two groups six months later with data on aging parameters for learning, strength, and agility when mice were 24 months of age. I found that mice with faster ear biopsy closures were better learners, and showed increased strength compared to mice with slower ear biopsy closures. I will be doing a special stain for collagen on formalin fixed ear wound areas to confirm the healing process and see if digital imaging staining intensity correlates with healing rate and aging parameters. These preliminary observations suggest that a simple skin biopsy procedure can be used to predict levels of resilience to aging phenotypes and identify mice at increased risk for age-related frailty conditions.

The concept of mild cognitive impairment (MCI) has evolved over the past several decades to represent a state of cognitive function between that seen in normal aging and dementia. Clinically, it is challenging to fully characterize and propose strategies for treatment partly becuase little is known about the extent of underlying pathology and why some individuals are resistant, and some are susceptible. This study describes a model of naturally occurring MCI in middle-aged mice that will help address neuropathology and resistance questions. C57BL/6 mice, 20 months of age, were tested for cognition using a spatial navigation learning task, a box maze, that identified a fast-learning group (MCI resistant) and a slow-learning group (MCI susceptible). Regarding strength and activity performance, both groups performed equally well. Brains were collected and either flash frozen and stored at -80°C, or placed in formalin for sectioning and immunohistochemistry (IHC). Preliminary data suggest a number of messaging pathway differences in the brains of MCI resistant and MCI susceptible mice. One such pathway appears to involve inflammatory cytokines providing the rationale to test for expression of inflammatory regulators MCP1 and TNF-𝜶, using IHC and digital imaging. I am currently staining the brains using these IHC assays and then running the images through a digital imaging program known as Qupath. The expectation is that MCI resistant mice will display low levels of MCP-1 and TNF-𝜶 expression while MCI susceptible mice will show high levels of MCP-1 and TNF-𝜶 expression in the brain. Additional data will help establish naturally occurring cognitive impairment in middle aged mice as a useful model for studying neuropathology and therapeutic approaches related to MCI in people with increasing age.