Abstract 187: Trastuzumab induced myeloid cell alterations identifies mechanistic properties of vascular normalization
Authors: Bloom Meghan J. , Jarrett Angela M. , Triplett Todd A. , Syed Anum K. , Yankeelov Thomas E. , Sorace Anna G. 
Published: August 2019

Abstract
Introduction: The purpose of this study is to quantify longitudinal changes in myeloid cell infiltration after trastuzumab treatment to reveal mechanistic properties of improved tumor vascularization in human epidermal growth factor receptor type 2 positive (HER2+) breast cancer. We have previously presented in vivo data showing that trastuzumab increases vascular maturation leading to better delivery of therapeutics. The balance of myeloid cells in the tumor microenvironment (TME) can impact neovascularization. Specifically, M2 macrophages (MACs) promote angiogenesis causing irregular vascularization and M1 MACs exhibit anti-angiogenic properties counteracting the M2 cells and improving tumor perfusion. We present results revealing macrophage polarization towards an M1 phenotype after trastuzumab therapy in HER2+ tumors.
Experimental Design: 107 BT474 breast cancer cells were implanted subcutaneously into athymic nude mice. After tumors reached 250 mm3, mice were treated with trastuzumab (10 mg/kg) or saline over one week and were excised for analysis on days 0, 4 and 7. Half of each tumor was fixed in 10% neutral buffered formalin for immunohistochemical staining (IHC) of the TME and half was digested for analysis of myeloid cell infiltration with flow cytometry. The flow cytometry panel used consists of ten markers, two specifically for the identification of M1 and M2 MACs; CD38, and CD206, respectively. A non-parametric Wilcoxon rank sum test was used to determine statistical differences between control and treated groups.
Results and Discussion: Initial studies revealed an increase (P = 0.03) of MACs in day 4 and day 7 treated tumors. Of these MACs, there was an increase (P = 0.03) in co-expression of M2 and M1 phenotype markers CD206 and CD38 in day 4 treated tumors indicating a phenotypic switch occurring. After a third dose of trastuzumab, there was an increase (P = 0.03) in M1 MACs (CD38+/CD206-) in day 7 treated tumors. In day 4 and 7 treated tumors, there was a decrease (P = 0.02) in non-differentiated MACs (CD38-/CD206-) supporting evidence that a phenotypic switch is occurring. Ongoing studies are evaluating spatial variations in MAC infiltration (CD206, F4/80), hypoxia (pimonidazole), and vascular maturation index (ratio of alpha-smooth muscle actin to total vessel counts as stained with CD31) through IHC. Cytokine detection assays are being done to evaluate changes in cytokines contributing to macrophage polarization and angiogenesis.
Conclusion: These results offer strong evidence supporting the hypothesis that MACs in the TME are switching to an M1 phenotype after trastuzumab treatment and contributing to trastuzumab induced vascular alterations. Analyses of IHC will validate vascular changes occurring simultaneously with myeloid infiltratration.
Citation Format: Meghan J. Bloom, Angela M. Jarrett, Todd A. Triplett, Anum K. Syed, Thomas E. Yankeelov, Anna G. Sorace. Trastuzumab induced myeloid cell alterations identifies mechanistic properties of vascular normalization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 187.