COLORECTAL CANCER PRIMES LIVER RECRUITMENT OF CCR2+ TUMOR ASSOCIATED MACROPHAGES TO ESTABLISH A PRE-METASTATIC NICHE
Timothy Nywening, Andreas Karachristos*
University of South Florida, Tampa, FL
Introduction: Colorectal adenocarcinoma is one of the most common causes of cancer related mortality globally. The liver is the most common site of distant metastasis with 25% of patients presenting with synchronous liver disease at initial diagnosis. The mechanisms by which the colorectal liver metastasis microenvironment is established is currently not fully elucidated but involves an intricate interaction among metastatic colorectal cancer cells, the inflammatory immune infiltrate, and liver parenchyma. In particular the role of tumor associated macrophages (TAM) expressing the chemokine receptor CCR2 have been shown to play a prominent role in multiple malignancies and targeting the CCL2/CCR2 chemokine axis is a promising strategy in early phase clinical trials. In this study, we examine the role of CCR2+ macrophages in facilitating the establishment of a pre-metastatic niche in colorectal cancer.
Methods: Animal studies using the MC38 murine colorectal cell line labeled with luciferase were conducted using a reliable orthotopic model of colorectal hepatic metastases previously described (Grossman & Nywening, et al. 2018). Subjects were selected following confirmed absence of detectable established colorectal liver metastases via bioluminescence imaging and pathologic assessment. Livers were perfused to eliminate peripheral blood contamination and specimens allocated for analysis by flow cytometry, quantitative real time PCR, and immunohistochemistry. Statistical analysis was conducted using GraphPad Prism software with a p-value <0.05 considered significant.
Results: Using a preclinical murine model in animals with established tumor, the liver pre-metastatic niche was examined. Analysis revealed an increase in CCL2 production by the pre-metastatic liver in tumor bearing mice (Fig 1A: p<0.01). This correlated with a significant increase in CCR2+ tumor associated macrophages (TAM) relative to controls by flow cytometry ( Fig1B: 2.2% vs 1.1% of total cells; p=0.02). This influx of TAM was associated with elevated hepatic vascularity, demonstrated by an increase in CD31+ endothelial cells compared to the livers of non-tumor bearing animals (3% vs 1%; p=0.04). Furthermore, characterization of the tumor infiltrating lymphocyte (TIL) population found an abundance of immunosuppressive FoxP3+ regulatory T-cells with a paucity of ant-tumor effector CD8+ TIL.
Conclusions: These results suggest recruitment of TAM to the pre-metastatic liver in colorectal cancer prior to detectable established tumor. This occurs concurrently with an increase in vascularity and establishment of an immunosuppressive microenvironment. Further exploration of these findings may help elucidate potential mechanisms of metastatic spread in colorectal cancer and the development of novel treatment strategies.
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