The poor survival, high prevalence of smoking and alcohol use, and advanced stage at diagnosis of esophageal squamous cell carcinoma (ESCC) in the VA population necessitates further study into new management strategies for squamous cell carcinoma. Over the past few years, emerging immunotherapy approaches, such as adoptive cell transfer, have highlighted the importance of understanding and manipulating the tumor microenvironment. The tumor microenvironment in ESCC is understudied. In this proposal, we focus on determining the role of eosinophils, a key component of the microenvironment, in ESCC. We have generated preliminary data demonstrating the presence of eosinophils in human ESCC tumors but not in tumor-adjacent tissue. We then established and characterized the 4-nitroquinolone-1-oxide (4-NQO) murine model of as a feasible model for determining the function of eosinophils in the tumor microenvironment. We show that, in the 4-NQO model, eosinophils are constantly being produced by the bone marrow, recruited specifically to areas of esophageal dysplasia as in human ESCC, and not present in areas without dysplasia. Additionally, Eotaxin-1 (CCL11), a potent eosinophil chemoattractant, is induced in areas of dysplasia. These observations suggest that eosinophils play a tumor specific role and are not the result of a more general inflammatory response. While the 4-NQO model will be employed to generate a broader understanding of eosinophils? role in tumorigenesis, sophisticated co-culture techniques the we have developed provide mechanistic insights into eosinophil activation and function in tumor biology. In this proposal we show that eosinophils co-cultured with esophageal cells increases apoptosis, decreases viability, and alters the cytokine profile of epithelial cells, increasing expression of TNF-a and IL-2, IL-8, and IL-10. Furthermore, eosinophils, which produce reactive oxygen species (ROS), have increased expression of eosinophil peroxidase after co-culture with epithelial cells. This is significant as eosinophil peroxidase is known to catalyze the oxidation of halides and thiocyanate, resulting in the release of cytotoxic reactive oxidant species. Taken together, these data suggest that eosinophils may decrease viability ESCC cells via release of ROS. Glutathione Peroxidase 1 (Gpx1), an abundant antioxidant enzyme expressed by eosinophils, catalyzes the reduction of hydrogen peroxide or lipid peroxidases to water. Here we independently propose that Gpx1 regulates ROS production by eosinophils, and the decrease in expression of Gpx1 by eosinophils after co-culture with esophageal epithelial cells suggests it is modified by the eosinophil- epithelial interaction. In light of these data and the recruitment of eosinophils specifically to areas of dysplasia and carcinoma in ESCC, our overarching hypothesis is that eosinophils are cytotoxic to tumor cells and thus are protective in ESCC.
The specific aims for this project are: 1. To determine the role of eosinophils in ESCC tumorigenesis. We will utilize the 4-NQO model of ESCC and compare esophageal tumorigenesis in Ccl11-/- versus WT mice after treatment with 4-NQO. We will then determine the impact of eosinophil depletion and hyper-eosinophilia on WT mice treated with 4-NQO at different time points. 2. To define the effect of eosinophils on epithelial signaling and homeostasis. We will determine the effect of eosinophils on normal and dysplastic epithelium by co-culturing eosinophils with mouse organoids. More specifically, we will define the role of Gpx1 on eosinophil function and activation by co-culturing Gpx1-/- eosinophils with both wild-type organoids and tumoroids. These studies have the potential to improve understanding of eosinophil biology within the context of ESCC, which could lead to specific therapeutic interventions targeting eosinophil function.
Esophageal Squamous Cell Cancer (ESCC) accounts for approximately 90% of the incident 456,000 esophageal cancers worldwide. Data from the VA Central Cancer Registry (VACCR) between 1995-2005 revealed 3,493 new cases of ESCC (44% of all esophageal cancers). Patients often present with advanced disease, as 60-91% of patients presented with at least Stage II disease, and 26% present with metastatic disease. Current therapies include a combination of chemotherapy, radiation, and surgical intervention, which are not specific and in many cases not effective. Median survival time after diagnosis is 14.2-15.3 months. As such, new, more specific therapies are necessary. The advent of immune checkpoint inhibitors to reactivate antitumor immunosurvelliance and clearance represents a new treatment avenue for difficult to manage cancers. Our overarching hypothesis is that eosinophils could be cellular targets for immunotherapy in difficult to manage patients with ESCC.
