Abstract P1-03-08: In vivo investigation of the Duffy antigen receptor for chemokines (DARC/ACKR1) and tumor-infiltrating immune cells in the breast tumor microenvironment
Authors: Martini Rachel , Newman Lisa , Manley Nancy , Davis Melissa 
Published: March 2020

Recruitment of immune cells and subsequent infiltration of these cell populations into the tumor microenvironment (TME) can influence disease prognosis, and potential treatments for patients. Through studying the TME of breast tumors, we have investigated factors that modify immune cell recruitment and infiltration into the tumor area. In our recently published work, we analyzed the TCGA breast cancer (BC) cohort, where we have identified the Duffy Antigen Receptor for Chemokines / Atypical Chemokine Receptor 1 (DARC/ACKR1) as a putative driver of immune cell recruitment and infiltration to the breast TME.

DARC/ACKR1 is an atypical chemokine receptor that promiscuously binds both the CC and CXC classes of chemokines. On the surface of red blood cells in circulation, DARC/ACKR1 maintains homeostatic levels of chemokines in circulation by binding chemokines when levels are high, and releasing them back into circulation when levels lower. In tissues, DARC/ACKR1 participates in chemokine transcytosis, working to move chemokines across the cell layer to establish chemokine gradients necessary for recruitment of respective immune cell populations.

Through in silico analysis of TCGA BC cohort data, we performed CIBERSORT TME deconvolution methods to approximated levels of 22 tumor-associated immune cell populations. We showed that as DARC/ACKR1 expression increased in breast tumors, we also saw a significant increase in number of tumor-associated immune cells (p&lt;0.0001). When looking at the specific cell populations, we found that B cells, T cells, monocytes and macrophage populations were significantly different between high, mid and low DARC/ACKR1 expressing tumors.

To continue investigation of these findings in vivo, we developed a novel DARC/ACKR1 breast cancer transgenic mouse model. Briefly DARC/ACKR1 -/- mice were crossed with the C3(1)Tag breast cancer transgenic mouse model. Breast cancer Tg+; DARC/ACKR1 +/- males were then backcrossed to DARC/ACKR1 -/- females, to generate target female mice that were BC Tg+, and either DARC/ACKR1 +/- or DARC/ACKR1 -/-. Disease progression in the C3(1)Tag line follows a similar pattern to ductal carcinoma in situ (DCIS), through progression to advanced invasive disease. All mice develop DCIS beginning at approximately 12 weeks of age, and 100% of mice are affected with invasive disease by 6 months of age.

We have collected tissue from mice from DCIS stage of disease through invasive disease. Blood was collected from each mouse, and the plasma was used to quantify the circulating chemokine profile. Tumors and mammary tissue were collected from each mouse and weighed, alongside spleen, liver, and potential metastatic tumor sites. Tumors and mammary glands were processed for immunohistochemistry and immunofluorescent staining procedures, where DARC/ACKR1 expression, immune cells, structural markers and chemokines of interest were assayed. We also took representative sections from each genotype of interest for Hyperion imaging mass cytometry analysis, where we can simultaneously analysis 27 different immune, structural and other markers of interest. From our preliminary results from in silico TCGA analysis, we expect to see changes in immune cell recruitment and infiltration, specifically in quantity of immune cells recruited and infiltrating, and in the composition of the tumor associated immune cells between our DARC/ACKR1 expression or non-expressing BC Tg+ mice.

Citation Format: Rachel Martini, Lisa Newman, Nancy Manley, Melissa Davis. In vivo investigation of the Duffy antigen receptor for chemokines (DARC/ACKR1) and tumor-infiltrating immune cells in the breast tumor microenvironment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-08.