Abstract P4-06-17: Investigating trastuzumab-induced myeloid cell alterations for improving combination therapy in HER2+ breast cancer
Authors: Bloom MJ , Jarrett AM , Triplett TA , Syed AK , Yankeelov TE , Sorace AG 
Published: May 2019

Introduction: The purpose of this study is to identify temporal changes in the identity and cellularity of myeloid cells infiltrating human epidermal growth factor receptor type 2 positive (HER2+) breast cancer following trastuzumab therapy. Trastuzumab is a targeted therapy used in combination with cytotoxic therapies to treat HER2+ breast cancer. Previous data shows that in HER2+ tumors, trastuzumab increases vascular maturation and decreases hypoxia, both of which can sensitize tumors to cytotoxic therapies. Preliminary immunofluorescent data reveals a significant increase in the amount of myeloid cell infiltrates in trastuzumab treated tumors during the same temporal window as vascular and hypoxia alterations. The balance of myeloid cells in the tumor microenvironment (TME) can impact neovascularization, hypoxia, and tumor progression. Thus, we hypothesize that quantifying immune modulation following trastuzumab treatment will identify mechanistic properties of trastuzumab induced vascular alterations in the TME. We present results identifying an increase in various myeloid infiltrates between control and trastuzumab treated tumors in HER2+ breast cancer.

Experimental Design: BT474 HER2+ breast cancer cells were implanted subcutaneously into athymic nude mice. After reaching 250 mm3, tumors were treated with trastuzumab (10 mg/kg) or saline for one week and were excised for analysis on days 0, 4, and 7. Each tumor was cut at the largest cross-section; half was fixed in 10% neutral buffered formalin for immunohistochemistry staining of TME markers (pimonidazole, CD11c, F4/80, CD31, and alpha-SMA), and half was digested for single cell analysis using flow cytometry. Data was analyzed with FlowJo software. Distinct populations of myeloid cells have been quantified in day 4 control (n = 6) and trastuzumab treated (n = 4) tumors. A non-parametric Wilcoxon rank sum test was used to determine statistical differences.

Results and Discussion: Flow analysis revealed a significant (p &lt; 0.05) increase of macrophages in tumors treated with trastuzumab for 4 days compared to control tumors. Spatial immunofluorescent data confirms these findings showing a significant increase in macrophage populations, indicated with increased CD11c+ and F4/80+ co-staining in day 4 treated tumors compared to control (p &lt; 0.01). Additionally, flow cytometry results show a significant (p &lt; 0.05) increase in neutrophils in day 4 trastuzumab treated tumors compared to control tumors. Ongoing studies are evaluating spatial variations in myeloid infiltration (CD11c, F4/80), hypoxia (pimonidazole), and vascular maturation index (ratio of alpha-smooth muscle actin to total CD31 stained vessel count) through immunohistochemistry as well as comparing myeloid infiltration in trastuzumab-treated and control mice on day 7.

Conclusion: Quantifying trastuzumab induced immune-modulation will provide mechanistic insight into how trastuzumab alters the TME and improves tumor sensitivity to treatment. This information will be of great importance to identifying optimal windows to administer combination therapies as well as in the development of novel immunotherapies in breast cancer.

We acknowledge the support of CPRIT RR160005, NCI R01CA186193, and ACS RSG-18-006-01-CCE.

Citation Format: Bloom MJ, Jarrett AM, Triplett TA, Syed AK, Yankeelov TE, Sorace AG. Investigating trastuzumab-induced myeloid cell alterations for improving combination therapy in HER2+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-17.