Abstract C19: The role of resident liver macrophages in suppressing the progression of hepatic micrometastases from pancreatic ductal adenocarcinoma

Background: The majority of patients with localized pancreatic ductal adenocarcinoma (PDAC) die from metastatic disease, typically of the liver, despite a margin-negative (R0) resection. Therefore, these patients likely harbor occult metastatic disease in the liver at the time of surgery. In this study, we evaluated the role of resident liver macrophages in suppressing the progression of hepatic micrometastases using a murine model of micrometastatic PDAC with patient-derived xenografts (PDXs). Methods and Results: Low-passage, patient-derived KRAS-mutant tumor cells expressing firefly luciferase were injected into the spleens of athymic nude mice resulting in liver metastases, followed by splenectomy to remove the primary tumor. Hepatic tumor burden and metastatic growth kinetics were evaluated by bioluminescent imaging on post-injection days 1, 2, 3, 7, and weekly thereafter. Each of the PDX tumors exhibited a decline in tumor burden over the initial days after injection followed by a period of quiescence with a reproducible, PDX-specific time to proliferative outgrowth ranging from 15 days to greater than 200 days. To assess the role of apoptosis in initial tumor cell clearance and suppression of outgrowth, mouse liver preparations were analyzed for expression of cleaved caspase-3 and cleaved PARP in tumor cells using flow cytometry. There was low expression of both apoptosis markers, suggesting that apoptosis is not a major cell-clearance mechanism. Athymic nude mice lack cell-mediated immunity but have functioning innate immunity. Because there is a large population of resident macrophages in the liver, we hypothesized that macrophages play an important role in the clearance and suppression of hepatic PDAC metastases. To test this, hepatic metastasis outgrowth was assessed following macrophage ablation with liposomal clodronate treatment of mice 48 hours prior to tumor cell injection. Following macrophage ablation, there was a trend toward less robust initial tumor cell clearance and a significantly decreased time to proliferative outgrowth compared with control (13 days vs 26 days, p=0.039). H&E sections demonstrated an abundance of hepatic macrophages surrounding tumor cells in the control group while this response was absent in the clodronate group. Splenic tumor cell injections were repeated using athymic nude mice vs NOD scid gamma (NSG) mice which lack both cell-mediated immunity as well as functional macrophages, dendritic cells, and NK cells.