Abstract P009: Correlation between immune modulation of macrophage recruitment and new blood vessel formation in a subcutaneous murine mouse model of colorectal cancer
Authors: Bess Shelby N. , Muldoon Timothy J. 
Published: January 2022

Abstract
Purpose: Colorectal cancer is the fourth most common cancer in the United States, accounting for more than 50,000 deaths annually1. The standard treatment is 5-fluorouracil (5-FU)-based chemotherapy, but emerging methods of immunotherapy have gained the attention of investigators and clinicians. A specific cytokine-based strategy for immunotherapy is the blockade of CCL2/MCP-1 (monocyte chemoattractant protein-1)2. CCL2 is a chemotactic cytokine that recruits circulating monocytes to the tumor microenvironment2. These monocytes differentiate into tumor-associated macrophages (TAMs), which promote pro-tumor functions including tumor growth, immunosuppression, and angiogenesis. CCL2 blockade in certain mouse models of cancer has been shown to reduce tumor burden but had not been studied in colorectal cancer. In this study, we examine how cytokine-targeted anti-CCL2 immunotherapy affects tumor-associated macrophage recruitment as well as the promotion of angiogenesis as a standalone and in combination with 5-FU.
Methods: Nine-week-old Balb/c mice (n=13) were acclimated for a week in the Small Animal Facility at the University of Arkansas. CT26 cells, a murine colon carcinoma cell line, were cultured in RPMI-1640 media, supplemented with 10% bovine serum, 0.2% amphotericin B/gentamicin, and 1% antibiotic antimycotic solution. 1 × 106 CT26 cells were subcutaneously injected into the left flank of Balb/c mice (10 weeks old). The tumors grew to 75 mm3, upon which Balb/c mice were randomly divided into saline control, immunotherapy, chemotherapy, and combination (immunotherapy + chemotherapy) groups. The immunotherapy and chemotherapy groups were intraperitoneally injected with a 28G insulin syringe with anti-CCL2 and 5-FU at a concentration of 5.0 mg/kg/dose and 15mg/kg/dose, respectively. The control group was injected with an equal volume of sterile saline on days while the combination group received a dose of anti-CCL2 and 5-FU. Tumor-associated macrophages were quantified via immunohistochemistry using anti-CD68 and tumor vasculature was quantified using anti-CD31. Correlation plots were also created to determine a relationship between macrophage recruitment and new blood vessel formation.
Results: There was a decrease in the total number of tumor-associated macrophages from Day 3 to 7 (33% decrease) when tumors were treated with anti-CCL2 and 5-FU. The area of vasculature in the combination group showed a slight decrease from Day 3 to Day 7 (29% decrease). There was a slight positive correlation between total macrophage recruitment and new blood vessel formation (R2 = 0.3041).
Conclusion: Overall, we have demonstrated that anti-CCL2 in combination with 5-FU has modestly slowed tumor growth, reduced the area of vasculature, and decreased the number of recruited tumor-associated macrophages in a murine model of colorectal cancer.
Acknowledgments: This material is based on work supported by the National Institutes of Health (1R15CA202662- 01), National Science Foundation CAREER award (CBET 1751554), and the Arkansas Biosciences Institute.
Citation Format: Shelby N. Bess, Timothy J. Muldoon. Correlation between immune modulation of macrophage recruitment and new blood vessel formation in a subcutaneous murine mouse model of colorectal cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P009.